Alexandre Surrallés

Chromatic Conversations in Candoshi A Reply and Some Further Reflections

EN QUESTION

L'HOMME 230 / 2019, pp. 105 à 116

In an article entitled « On Contrastive Perception and Ineffability : Assessing Sensory Experience Without Colour Terms in an Amazonian Society » [START_REF] Surrallés | On Contrastive Perception and Ineffability : Assessing Sensory Experience Without Colour Terms in an Amazonian Society[END_REF], I affirmed that the Candoshi (an Amerindian people of horticulturists, hunters and gatherers of the Peruvian High Amazon) neither have in their language a term to name the concept of colour nor nomenclature to refer to specific colours. This data, corroborated in different specific surveys on colours that we have carried out among the Candoshi since 1998 in addition to the several years of fieldwork during the first half of the 1990s, contradicted the data presented in The World Color Survey (Wcs) (Kay et al. 2009 : 155-157). Our conclusions thus added to the list of works by linguists and anthropologists who observe the absence of colour names in languages around the world 1 , some of which also appear in the Wcs examples. The main author of the Wcs, Paul Kay, has questioned my data in a critical article (2018) with some unconvincing arguments, as I will demonstrate in the text that follows. However, the main objective of my article was not to present these facts that question Wcs data, but rather a very different one. So, before addressing Kay's criticism, we would like to summarise briefly the purpose of the article.

After framing my research in the area of what has been called studies on « ineffability » [START_REF] Levinson | Differential Ineffability and the Senses[END_REF] and after demonstrating that the Candoshi language does not have colour names, the main objective was to explore how the Candoshi can talk about colours without terms to name them. Detailed surveys of how Candoshi refer to the colours of objects allowed me to describe a technique I call « contrastive perception », which is used not only to talk about colours without specific terms for these, but also to evaluate the perception of things, in general, without any classic procedure of categorisation. This technique called « contrastive perception » has three fundamental characteristics, which I call comparison, context and instability. In short, and as far as colour is concerned, this technique is summarised in using a comparison to describe the colour of an object. For example, the colour of toucan feathers is like the fruit of the achiote tree (Bixa orellana). This comparison is always contextual, since without colour terms, the colour of things is not intrinsic to them and, therefore, not stable, since it depends on environmental circumstances in relation, for example, to luminosity. This is how the feathers, similar to the fruit of the achiote tree, can, in less luminous circumstances, be similar to the colour of blood and, in even less light, to the fruit of the hungurahua palm (Oenocarpus bataua). Indeed, as the comparison and the context introduce an element of contingency between the referent and the referenced, and therefore, indetermination, a large space for creativity appears from the combination of the multiple perceptive facets of the compared objects such as the polysemy of the references, allowing for the establishment of chains of very expressive analogies. Furthermore, since there is no rigid fixation in Candoshi between words and referents, there is no way to establish which of the terms corresponds to each object and therefore which is the original and which is the one compared. This anti-substantialist approach to the object during its description shows that there is no origin : these references are circular, working in a network, without an initial definition. This leads me to conclude that : « Meaning in context, comparing and using the relationship between words and objects in a non-stable and creative manner, as appears to be the case in the mode of perception that we have termed "contrastive", could express a particular way of treating sensory experience or precepts. Whereas conventional wisdom considers perception as a vertical relationship between the treatment of sensory data and the semantic identification of the category, in an ascending or descending direction, contrastive perception seeks to establish a horizontal relationship between labelled packages of sensory experience, as we have described. Each entity of the world can thus be described by a criterion context-dependent that, by the very dynamic nature of these criterions, will change of nature while described in relation to others » (Surrallés 2016 : 975, see also Surrallés 2017).

Kay is not interested in these propositions to which he says he has nothing to add or subtract. He only aims to question my data related to the absence of names of Candoshi colours, according to the terms defined by the Wcs. For me, the article is an indivisible piece. The two parts into which Kay divides it are intimately linked in the demonstration of the general proposition and one part cannot be explained without the other. The extrapolation of my text made by Kay seems to me problematic and unjustified at first glance and I believe it invalidates his entire criticism. Nonetheless, I will proceed to respond to his arguments point by point, beginning with the introductory methodological question of Kay's response and then the three « facts » of my article that Kay considers wrong.

Methodological Issues

Paul Kay's text begins with a reference to the methodology of the survey Wcs uses, which is questioned in my article. In essence, Kay criticises my claim that the method used both in the Wcs, and in the programmatic previous work of Paul Kay and Brend Berlin in Basic Colors Terms (Bct) (1969 : 1-14), establishes a list of colour terms before asking the informants about the scope of each of these terms on the colour palette. Kay states that this is not true and that a correction of the methodology used in Bct was made in the Wcs. Let's see if this is accurate. In the Bct, the methodology pre-established these basic colours that the interviewers had defined themselves, to the extent that they knew the language or by talking to the informant. The data were gathered in two stages. First, the basic colour words of the language in question were elicited from the informant, using as little as possible of any other language. Secondly, each subject was instructed to map both the focal point and the outer boundary of each of his basic colour terms on the array of standard colour stimuli described above (Ibid. : 5).

The survey methodology carried out in the Wcs attempted to correct this point, which obviously skewed the whole survey and predetermined its result, in order to overcome this naive error of misconception. But does the new Wcs methodology really overcome this problem ? The new Wcs methodology adds an additional, prerequisite stage called « Naming Task » to the single Bct process in order to correct the problem. This additional stage of the Wcs consists of first determining, with a supposedly objective method, the names of the basic colours in the language examined from each of the 330 individual colour stimuli, « one by one, according to a fixed random order, and a name was elicited for each » (Kay et al. 2009 : 13). Now, if we look at the document « Instructions to Field Workers » (Ibid. : 585-592 and the link presented by Kay 2018 : 96) signed by Brent Berlin, Paul Kay and Willian Merrified of the Summer Institute of Linguistics (Sil), we realise that the efforts made in the Wcs cannot overcome the flagrant problem presented by the Bct methodology. They do not overcome the difficulty because the survey discards any response from the informant that does not fit what the results want to show : that is, that there are names of colours in that language and that there are also some colours considered « basic ». I will not expand on this point. I think it is enough to reproduce the titles of these instructions : « A Note of the Kind of Response We Are Looking For » and « Defining the Concept Basic Color Term » (Kay et al. 2009 : 587). These instructions, as their titles themselves indicate, allow for free interpretation on the part of the Field Worker (I suppose a Sil missionary linguist) who can choose only those answers by the informant which fit the idea of a « Basic Color Term » that is sought-after, in such a way that the problem the Bct methodology attempted to correct is reproduced again in the Wcs. Of course, nothing in either the Bct or the Wcs methodology demonstrates that for a given language there is a notion of colour and words to name the colours considered basic. The idea that in every language there are names of basic colours is a prior premise for these studies and is not subjected to any empirical verification.

The Three Facts

After this methodological clarification, Kay's article tries to refute the three facts that I introduce to show the weakness of the data presented by the Wcs related to the supposed terms for Candoshi colours and to conclude that, in reality, there are no names for colours in this language.

The First "Fact " « Terms that the Wcs has identified as Candoshi colours cannot be considered true terms, that is monolexemic or morphologically simple terms for colours, as Berlin and Kay claim (1969 : 6). They are more complex syntactic constructions. For example, ptsiyaromashi, the term for "yellow", is constructed by using two morphemes, ptsiyaro/ mashi, which means milvago/like, according to the Candoshi dictionary (Tuggy 1966 : 64). The translation would be "like or similar to the feathers of a Milvago bird". Another example, kantsirpi, was the term chosen to name the colour "black" and is composed of three parts, kansi/ar/pi, that mean tar/have/towards, something like "is similar to tar", according to the dictionary (Ibid. : 26) » (Surrallés 2016 : 966).

To dispute this « fact », Kay says in his reply that the terms ptsiyaromashi or kantsirpi are actually « derived words » (2018 : 98). This only confirms my suspicion that it is not about « monolexemic » terms as would be necessary according to the definition that he proposes of the « concept of basic colour terms » (Berlin & Kay 1969 : 5-6). Kay says that these terms are derived words, similar to the English words « fishy », « horsey » or « sheepish » (2018 : 94) and therefore they are not a « syntactically structured phrase» but a word, a derived adjective. Against this affirmation it could be said that it is difficult to determine in an agglutinating language such as Candoshi the boundary between word and phrase. It is easier to determine when a word is monolexemic or when it is not, and several Candoshi terms for colour in the Wcs are not monolexemic. However, in addition, terms similar to these are explicitly questioned in rule 1 of the definition of the original notion when Kay states that derived adjectives such as « bluish » or « lemon-coloured » (among others) cannot be considered basic (Berlin & Kay 1969 : 5-6). Moreover, rule 6 considers the terms for colours that « are also the name of an object characteristically having that colour » as the two Candoshi terms in question. In relation to this fact, Kay concludes with a somewhat paradoxical critique when he says that even if we consider that these two terms are « polymorphemic » (2018 : 95), that is, they contradict rule 8 of the concept of basic colour according to which they must be « morphological simple » terms, this does not prove that the other colours of the list proposed by the Wcs do not conform to the basic colour criteria. So, contradicting any methodology of scientific testing that considers that a refuted case is sufficient to disprove the hypothesis, Kay believes that it is not enough for me to prove that two of the most important terms of the eight Candoshi terms for colour according to the Wcs do not conform to the concept of basic colour term to reject the set of data on Candoshi colours of the Wcs. We consider, on the contrary, that it is more than enough and, of course, we could add other terms like kavabana (a complex term formed by kavaro/tama, parrot/similar, that is « similar to the feathers of the macaw »), although it is not up to us to deny each of the terms, as we could do very easily, to question the whole.

The Second "Fact" « The supposed Candoshi colour terms are words or phrases that mean other things, but which have been regarded as terms only because of bias introduced by the linguist. Indeed, all the Wcs Candoshi colour terms refer to something other than colour. This can be verified by means of a simple experiment : if a Candoshi-speaker is asked what ptsiyaromashi or kantsirpi means, they will never answer "yellow" or "black", but make reference for instance to a bird and a resin (tar) used to coat peashooters and as fuel for torches » (Surrallés 2016 : 966).

Kay does not understand what I mean when I say that « if a Candoshispeaker is asked what ptsiyaromashi or kantsirpi means, they will never answer "yellow" or "black" » and supposes that I am referring to the answers of Candoshi people with sufficient knowledge of Spanish to use the terms « yellow » (amarillo) or « black » (negro). In fact, I was referring not only to Spanish, but also to Quichua, which is spoken as a regional lingua franca by several Candoshi or even the terms in English known by the Candoshi with some degree of school education. I refer to colour terms in other languages since I would have no other way to respond in their language that kantsirpi is the name of a colour, since they do not have a term to refer to the concept of colour. As confirmed by the linguist John Tuggy, contacted by Kay, there is no « lexeme that corresponds to the concept » (Kay 2018 : 103). This is for me the most important point on which I base my criticism of Kay's works. As other linguists have already pointed out repeatedly, if there is no term in the language for the concept of colour as a phenomenon, we cannot assume that this concept is conceived of by the speakers of this language in the same way as if it had a term assigned to it. It is only possible to presume its existence through the famous notion of covert category, that is, assuming that colour is an implicit category without nominalising. Now, the existence for the Candoshi of such a covert category of colour has not been demonstrated in the data presented in the Wcs. Nor does Kay demonstrate in his critical article that a term for colour implicitly exists in Candoshi. Neither does the linguist Tuggy, who claims that despite not having a lexeme for colour, Candoshi people « are very colour conscious » (Ibid.) without saying on what this impression is based.

Kay also claims that I do not present evidence of why the supposed Candoshi terms that have no-colour referents are not legitimate colour terms like the English orange or the French marron. For Kay, both the Candoshi and the English responses show that the terms for Candoshi or English colours are « notionally motivated » (Ibid. : 96). However, my entire article shows how the Candoshi people speak of colours through comparisons and this without needing to involve a notion of colour or a motivated colour nomenclature. This is significantly different than what occurs in European languages in which there is a very stable notion of colour and a typology of colours, learned since childhood. In any case, for a French or an English speaker, when they are asked what « orange » means, they will reply : a fruit and a colour, something that Candoshi people cannot do because of the lack of a term for colour. They cannot and they will not. And besides, they do not need to.

The Third "Fact"

« The list of Candoshi terms proposed by Kay, Berlin et al. has been arbitrarily put together whereas other expressions and terms emerge when Candoshi speakers are asked to name colours. In this respect, we interviewed a group of speakers, showing each of them the chips composing the Munsell chart used by Berlin and Kay, separately and randomly, and asked them to describe them to us in any way that came to mind […]. Indeed, the Candoshi used a different term or expression to respond to each chip that we showed them. The use of dozens of terms and expressions calls into question the selective data on the Candoshi reported in the Wcs » (Surrallés 2016 : 966-967).

Approximate scope on the Munsell colour chart of the most used Candoshi terms to name colours in my survey (adapted by the author from Surrallés 2016 : 966, 969) : boorshi (a), chobiapi (b), yobsa (c), kchomshi (d), koorashi (e), kachiva (f), ptsiyaro (mashi) (g), kantsirpi (h), goochi (i), ktsachi (j), kamachpa (k), tsarona (l), katama (m), kavabana (n), aroovi (o), tarika (p) and txobi (q) ; most of these terms refer to species or parts of animal or plant species Kay disputes my claim that the final list of alleged Candoshi colour terms is a fairly arbitrary selection made by the interviewer. He argues for his defence that the survey methods plan for a complex protocol -presented as an annex in the Wcs as we have already mentioned [START_REF] Kay | The World Color Survey[END_REF] : 585-592) -which reduces the arbitrariness and the bias potentially introduced by the interviewer. I have consulted the survey methods and, as I have stated in the section entitled « Methodological Issues » above, these seem very questionable to me, in particular, with regard to inducing the interviewer to an arbitrary selection of the answers considered « good », in that they do not contradict the thesis that they want to demonstrate about the universal existence of basic colour terms. In addition, the entire survey assumes the existence of colour names, and a local notion of the concept of colour, which is what many linguists and anthropologists question, working independently and in languages of all continents, as cited in the introduction.

Kay also reproaches me for not using quantitative analysis to reinforce my criticisms. He could even blame me for not using the survey method he recommends to reach the conclusions he is interested in. In fact, I do not present quantitative data because my data is not statistically relevant so as to deduce from it « unquestionable facts » about the Candoshi language. The problem is that what the Wcs presents, at least for the Candoshi people, does not have any statistical relevance either. With only 11 respondents (Ibid. : 155), less than half the number (25) required by the « Instructions to Field Workers » (Ibid. : 585), statistical data on the Candoshi language in general cannot be extracted. Presenting this data as relevant from a statistical point of view seems illegitimate. Neither the Wcs data nor my own data allow for quantitative analysis. However, this is not a problem for my position as I never intended to propose results based on statistics but rather on a qualitative basis supported by several field studies on the subject. The problem is present, though, for the Wcs, since all its methodology is based on the reliability of the data and the derived statistics.

Beyond Relativism versus Universalism

This debate suffers from an imbalance that Paul Kay tries to correct by involving the linguist John Tuggy who worked with the Candoshi people as a Sil missionary. In fact, Kay does not know the Candoshi language and I do not believe he has ever spent time among its speakers. For this reason, he asks Tuggy a series of questions, whose answers he adds at the end of the article. The problem for Kay is that the opinions of the linguist Tuggy confirm my criticisms of the Wcs data. Let us examine, for example, the translations in English that Tuggy now proposes in the appendix added by Kay (Kay 2018 : 102) for the Candoshi terms for colours, which according to him are more precise than the Spanish translations of his dictionary [START_REF] Tuggy | Vocabulario candoshi de Loreto[END_REF]. Not only do these more accurate English translations differ from the Spanish translations that he proposes in the same dictionary, but they also do not correspond to the English terms in the Wcs. One might even wonder if the English translation of the Candoshi colours now proposed by Tuggy and which includes terms such as « dark », « pale » and « light » can be considered English names of basic colours according to the conditions of the Bct and the Wcs. And the answer is that the said conditions would not admit these English terms as basic colour names. By this I only wish to point out that the question is much more complex than what Paul Kay's works would have us believe. The battery of questions that Paul Kay (2018 : 103) poses in a subsequent message by inducing John Tuggy's answers only confirm the distance between the Candoshi linguistic reality and the Bct and Wcs theory. Indeed, John Tuggy does not deny that these Candoshi terms are « morphologically complex », and the most important point is that he confirms my opinion, as indicated above, that there is no lexeme to refer to the concept of colour in this language. I would like to return to this point that Kay specifically addresses in note 9 of his text (Ibid. : 101). Kay, in this note, asks how, if the Candoshi people do not have a word for colour, as I have pointed out, they can say, as those from Bellona Island have said, « we do not talk much about colour around here » [START_REF] Kuschel | We Don't Talk Much About Colour Here" : A Study of Colour Semantics on Bellona Island[END_REF]. What the Candoshi people are expressing here, like the people of Bellona, is an ignorance of the concept of colour both in their language and in their practice. For this reason, colour is, for them, something difficult to identify with precision even though we attempted to explain it to them, using arguments and examples, even going so far as to refer to the meaning of the word « colour » in Spanish when the Candoshi interlocutor had some knowledge of the Spanish language. Kay also points out the following in his note : « Writers who suppose that lack of a word for colour in a language entails that the language can't have words and (conceptual categories) for colours seem to either ignore (e.g., Lucy 1997), or decline to consider (e.g., [START_REF] Wierzbicka | Why There Are no "Colour Universals" » in Language and Thought[END_REF], the existence of languages with words for "hot" and "cold" but no word meaning "temperature". According to Tuggy (2008) this is the case for Candoshi : ksani, "caliente", (hot/warm) ; kachízá, "frio" (cold/cool), no Spanish entry for "temperatura". Also Tuggy (2008) lists Candoshi ivaari [sic] "arriba" (up) and tsapoosho, "abajo" (down) with no gloss indicating "vertical direction" » (2018 : 101).

I will not insist on the difficulties of sustaining the idea of covert categories such as those proposed by Kay for temperature and vertical direction, based on simple common sense which, as we know, is the least common of the senses. I have already indicated the difficulty of using this concept citing the article by the linguist Anna Wierzbicka (2008 : 419), which is very convincing on this point, more so than the answer given by Kay and Kuehni in a note on this issue [START_REF] Kay | Correspondence[END_REF]. I would simply add that, without having carried out surveys for several years as for the colours (just as I did to obtain the results on the absence of Candoshi colors object of the debate), the paradigms that Kay establish for the Candoshi people regarding temperature or vertical direction seem to me very debatable. I will simply mention two pairs of antagonistic words in the dictionary. The terms ksani, « hot/warm » and kachizá, « cold/cool », do not exactly mean « hot/warm » and « cold/cool », because they do not apply to everything concerning the temperature. We can speak of a ksani or kachizá body as being hot or cold, but not of a hot or cold morning, for example. As for the pair ivaari, « up » and tsapoosho, « down », they would not indicate vertical direction because these terms refer to the area of thicket at the top of the trees, and to the ground respectively. For example, a bird or plane is not ivaari when they fly over trees and oil is not found tsapoosho before being extracted from the earth.

With this response to Kay's criticism I do not mean to say that works of a comparative nature, that seek to establish invariants of the human condition, are not possible. I believe they are desirable and necessary, and I wish to take this opportunity to express my admiration for the works of Paul Kay which I had the chance to read since my time as an anthropology student, despite disagreeing with the universalist thesis that inspires him. In fact, my own research aims to compare. It neither supports the universalist theses in relation to the nomination of colours, nor does it show opposing relativist theses. For us, this debate between universalists and relativists running through the recent history of anthropology has been exhausted, at least in terms of colour nomination. What we propose is to investigate, from a comparative point of view, the logic of difference in what might be called the perceptual act, to show how there are different ways, beyond classic categorization, to translate a sensation of colour into its nominalisation. Also, we want to demonstrate how these alternative ways can become as efficient, or even more efficient, if we take into account the type of information to be transmitted and to whom it is directed. To conclude, we will advance this last objective by way of example, in order to show the possibilities of this perspective of study.

Precision and Scope : Final Considerations

From classical Aristotelian logic to the recent prototype theory in cognitive science, there is a long intellectual tradition that marginalizes ways of describing sensations that do not entail explicit categorization. Indeed, a large number of scholars consider procedures that appear by default, in the absence of a simple categorical decoding system as ways to alleviate the lack of abstract thinking by means of solutions, sufficient to allow for communication, but of poor quality and effectiveness. Contrary to these opinions, we believe that these alternative routes would not always be a ratio difficilis, as Umberto Eco (1975 : 183) said, that is, ad hoc systems created when there is no possibility of communicating an experience through simple decoding (a ratio facilis). In the case, for example, of Candoshi contrastive perception, not only would it be a preferred way of communicating, chosen in the first instance, but it would also be a much more effective system of communication. In other words, the Candoshi prefer to use contrast, as I explained in my article (2016 : 975), rather than to name colours with any specific names. Moreover, they prefer it because it allows them to communicate better, communicate more and more easily. In order to explain what may seem like a paradox -that without names of colours we can communicate more and better about colours -we will need to present some considerations. In general, it is thought that a statement -depending on its brevity, accuracy and precision -communicates a certain idea more or less clearly, better or worse. From this point of view, it would be absurd to think that there could be a better way to describe objects than by using the words that exist to name them. For example, what could be better than the names of colours to describe chromatic tonalities? However, this idea doesn't take into account the fundamental relationship between the meaning that a statement may contain and its ability to be understood by a greater number of interlocutors.

To develop this idea, we will introduce two concepts : precision and scope. Let us assume that every statement aspires to possess at the same time the highest possible precision (describing the highest, most accurate number of qualities of an object with the lowest possible predicates), and the widest possible dissemination or scope (the ideas contained in the statements are understandable by the largest number of interlocutors). It turns out, nonetheless, that in general, precision and scope offset each other, so that the higher the precision, the more restricted the scope and vice-versa. Let us take colour as an example, so as to stay on-topic. The statement, « object X is red » is easily understood by all English speakers, although the information attached to the object is very weak ; it would be a statement with low precision, but wide scope. On the other hand, the statement, « object X is like a peach-palm fruit » specifies many characteristics of the object in question, among others, the hue of red, but presents a narrower scope because not every English speaker knows the fruit of the palm tree Bactris gasipaes. Taking into account the inversely proportional relationship between precision and scope, we can clarify the paradox referred to earlier when affirming that one can speak more and better about colours without specific colour terms. The technique that I have called contrastive perception allows us to describe colours without specific colour terms, in great detail, with efficiency and economy, that is, with great precision. However, it has a very low capacity for dissemination or scope because this communication technique based on comparison, contextualisation and contiguity requires the sharing of some lexical knowledge of very restricted scope, of a very particular ecological medium, both in a very profound way. Yet in the Candoshi context, the type of precise information conveyed by this procedure is very important, if not essential, for the type of data to be transmitted between interlocutors who share a very specialised and spatialised knowledge of the medium. In addition, there is practically nothing in their everyday environment that does not possess the colour with which it is naturally endowed. Consequently, there are no two identical things in different colours (or very few), contrary to what is usual in an urban context with access to a multitude of manufactured goods that are only distinguished by the hue.

Chromatic Conversations in CandoshiEN QUESTION


Keyu Li

Junke Wang

Birginia Espinoza

Yirui Xiong

Nan Niu

Jianxin Wang

Noelle Jurcak

Noah Rozich

Arsen Osipov

Mackenzie Henderson

Junke Wang

Jianxin Wang

Vanessa Funes

Melissa Lyman

Alex B Blair

Brian Herbst

Mengni He

Jialong Yuan

Diego Trafton

Chunhui Yuan

Michael Wichroski

Xubao Liu

Yuquan Wei

Lei Zheng
email: lzheng6@jhmi.edu.

Overcome the challenge for intratumoral injection of STING agonist for pancreatic cancer by systemic administration Authors

Keywords: pancreatic ductal adenocarcinoma, stimulator of interferon genes, innate immune agonist, STING, NLRP3, tumor microenvironment, immune checkpoint inhibitor, immunotherapy, systemic administration, intratumoral injection

Introduction

Recent research has highlighted the crucial role of the innate immune system in tumor immunosurveillance and the stimulation of antitumor immune responses [START_REF] Demaria | Harnessing innate immunity in cancer therapy[END_REF]. This has led to the development of several small-molecule innate agonists as potential immunotherapeutics or vaccine adjuvants for different types of cancer [START_REF] Rameshbabu | Targeting innate immunity in cancer therapy[END_REF]. However, despite promising preclinical studies, these agents have not been tested in most of tumor types due to the difficulties associated with their intratumoral delivery [START_REF] Mohseni | The function of cGAS-STING pathway in treatment of pancreatic cancer[END_REF]. Overcoming this challenge is critical for the clinical development of innate agonists. Nonetheless, the potential benefits of using innate agonists as T cell priming agents continue to provide an impetus for the exploration of novel strategies to overcome the challenge for their delivery.

Stimulator of interferon genes(STING) is a transmembrane protein that is expressed in various endothelial, epithelial and hematopoietic cells. Upon activation at the endoplasmic reticulum and subsequent translocation to the Golgi, STING recruits and activates TANKbinding kinase 1(TBK1), which in turn phosphorylates and activates interferon regulatory factor 3(IRF3) and NF-kB transcriptional programs, resulting in the expression and release of pro-inflammatory type I interferons(IFNs) and cytokines [START_REF] Barber | STING: infection, inflammation and cancer[END_REF][START_REF] Zhang | NF -κB signaling in inflammation and cancer[END_REF][START_REF] Kwon | The cytosolic DNA-sensing cGAS-STING pathway in cancer[END_REF]. Accumulating evidence has suggested that STING also possesses cell-intrinsic tumor suppressive activity [START_REF] Ni | T cell-intrinsic STING signaling promotes regulatory T cell induction and immunosuppression by upregulating FOXP3 transcription in cervical cancer[END_REF]. The mechanistic underpinnings of the cyclic GMP-AMP synthase-STING pathway make STING agonists(STING-A) a promising adjuvant to cancer vaccines [START_REF] Amouzegar | STING agonists as cancer therapeutics[END_REF]. Numerous natural or synthetic STING-A as monotherapy or in combined with other treatments have also been tested in both pre-clinical studies and clinical trials across many cancer types [START_REF] Naour | Trial watch: STING agonists in cancer therapy[END_REF][START_REF] Ding | Small molecules targeting the innate immune cGAS-STING-TBK1 signaling pathway[END_REF]. Synthetic cyclic dinucleotides(CDNs) were the first generation of STING-A that entered the clinical trial phase of drug development due to their structural versatility and ability to bind all prevalent allelic variants of human STING.

Pancreatic ductal adenocarcinoma(PDAC) is one of the most lethal malignancies due to its resistance to conventional therapies. PDAC and other non-immunogenic "cold" tumors do not respond to immune checkpoint inhibitors(ICIs) as monotherapy largely due to the lack of tumor-infiltrating effector lymphocytes [START_REF] Clark | Immunosurveillance of pancreatic adenocarcinoma: insights from genetically engineered mouse models of cancer[END_REF][START_REF] Koido | Current immunotherapeutic approaches in pancreatic cancer[END_REF]. Published studies have demonstrated that intratumorally injected STING-A inflamed the tumor microenvironment(TME) of PDAC with an effector T cell infiltration and reduced tumor burden in the mouse model of PDAC [START_REF] Jing | STING agonist inflames the pancreatic cancer immune microenvironment and reduces tumor burden in mouse models[END_REF]. However, despite these promising results at the preclinical phase, anti-tumor efficacies of STING-A have not been substantiated, largely due to the challenge of intratumoral delivery of such agents, which may have never reached therapeutic dose levels or never been tested in the appropriate disease indications. BMS-986301 is a novel CDN-based, next-generation STING-A and has demonstrated a promising antitumor activity in the CT26 and MC38 subcutaneous tumor murine models, resulting in more than 90% of tumor regression compared to only 13% with the firstgeneration STING-A, ADU-S100. Similar results were observed with a single intratumoral administration of BMS-986301 in combination with an anti-PD-1 agent [START_REF] Schieven | Preclinical characterization of BMS-986301, a differentiated STING agonist with robust antitumor activity as monotherapy or in combination with anti-PD-1[END_REF]. However, these previous studies were limited by the subcutaneously implanted tumors, which do not resemble the TME of human PDAC [START_REF] Vonderhaar | STING activated tumor-intrinsic type I interferon signaling promotes CXCR3 dependent antitumor immunity in pancreatic cancer[END_REF]. Therefore, this present study aims to deliver this next-generation CDN-based STING-A systemically and compare its anti-tumor efficacy and elicited immune response with the intratumoral injection of this agent in a liver metastasis model of PDAC.

Methods

Mouse experiments

The mouse study was conducted in accordance with the guidelines established by the Animal Care and Use Committee of Johns Hopkins University. Female C57Bl/6 mice aged 6 to 8 weeks were purchased from Jackson Laboratories and maintained under the Institutional Animal Care and Use Committee(IACUC) guidelines. Third-party management was responsible for maintaining the IACUC mouse protocol. STING-A(BMS986301, BMS) was dissolved into DPBS(Life Technologies) vehicle and administered to tumor-bearing mice either by intratumoral or intramuscular injection once a week at a dose of 5mg/kg starting on day 14, for a total of three doses. a-PD-1(BMS936558, BMS) and an IgG control(ab18443, BMS) were administered intraperitoneally twice weekly starting on day 14, for a total of five doses, at a dose of 10mg/kg.

NanoString

After the mouse was euthanized and the tumor was harvested, the tumor tissues were submerged into RNA-later(Invitrogen) to preserve the RNA. The total RNA was extracted from the whole specimen using the AllPrep DNA/RNA/Protein Mini Kit(Qiagen) according to the manufacturer's instructions. The RNA was then equalized for NanoString hybridization using the Formulatrix Tempest. The murine PanCancer Immune panel codeset XT_PGX_MmV1_CancerImm_CSO, Cat#115000142), which contains 750 target genes along with housekeeping and negative/positive control probes, was used for the NanoString hybridization. The data obtained was then analyzed using NanoString nSolver 3.0 and an internally developed NanoString Data Analyzer Rshiny app(BMS). The samples were run on the NanoString MAX system reader, and the data was analyzed using various CRAN and Bioconductor packages such as dplyr, tidyr, and reshape2 to clean, reformat and match the sample annotations to the normalized data exported from NanoString nSolverV.3.0.

Additional methods are provided in the Supplemental Materials.

Results

Develop a mouse model to resemble the intratumoral injection of STING-A in metastatic cancer patients

To examine whether systemic administration of STING-A is equivalent or superior to intratumoral(IT) injection, we established two mouse models to compare these two routes of innate agonist administration. We first developed a model to resemble the IT injection of STING-A in patients with metastatic diseases. This liver metastasis model was developed by implanting 7.5 x 10 5 tumor cells of the KPC tumor cell line, derived from the PDAC of the Kras/p53/pdx1-Cre(KPC) mouse model, through the hemisplenectomy procedure and splenic vessel injection to form liver metastases(Fig. S1A). The mice in this model would uniformly die from liver metastases if not treated [START_REF] Soares | A preclinical murine model of hepatic metastases[END_REF]. The changes in the sizes of the metastatic lesions in the liver can be monitored by small animal ultrasonography. Liver metastases have been identified as potential sites for the IT administration of the first-generation STING-A to treat patients with PDAC. In this study, the liver metastases serve as the clinically relevant target tumors for IT administration of STING-A(Fig. 1A).

Mice were also inoculated subcutaneously with the KPC tumor cell line to form subcutaneous(SubQ) tumors, not for the IT injection of STING-A, but for the abscopal effect to be examined. As shown in Fig. 1B, sixteen mice per group were treated by control vehicles(negative control, NC), a-PD-1, STING-A, and STING-A+a-PD-1(Combo). A total of five doses of a-PD-1 was administered twice a week by intraperitoneal injection(IP), while the STING-A was given for three weekly doses by IT injection to mimic local treatment or intramuscular(IM) injection to mimic systemic treatment. Mouse survival was followed; the target liver metastatic lesion was measured by ultrasound; and the subQ tumors was measured by calipers twice a week. We did not observe any obvious sign of toxicity including bleeding, infection, paralysis, and weight loss as a result of IT injection of STING-A.

However, we noticed small areas of liver necrosis around the injection sites at the necropsy of two mice(Fig. S1B). It should be noted that only approximately 35% of mice in the hemisplenectomy model harbored a liver metastasis feasible for IT injection.

The results(Fig. 1C) showed that the tumor growth inhibition(TGI) rate of the target liver metastasis lesion was significantly increased in the STING-A and a-PD-1 combination treatment group(maximum TGI=88.68±98.42%, p <0.05) and the STING-A group(maximum TGI=69.38±73.94%, p <0.05) as compared to the negative vehicle group. To investigate whether the local intratumoral injection of STING-A could induce the abscopal effect, we implanted subcutaneous tumors on the bilateral flanks of the liver metastasis mice model to mimic distant metastases. The TGI rate of bilateral SubQ tumors was also significantly increased in the combo group(maximum TGI=66.38±86.00%, p <0.05) as compared with negative control(Fig. 1D). Interestingly, the TGI in the distant SubQ tumors is bigger than that of the locally targeted liver metastatic lesion, supporting an abscopal effect from the STING-A and a-PD-1 combo treatment. Moreover, we compared the survival of liver metastasis mice in the above treatment groups and found that the combo treatment significantly prolonged survival comparing to the control treatment and the STING-A treatment(Fig. 1E).

Nevertheless, the combo treatment prolonged survival modestly without a statistical significance comparing to the a-PD-1 treatment. It is possible that the local intratumoral injection of saline may have caused inflammatory response which resulted in a small effect on priming the tumor for the a-PD-1 treatment. Nevertheless, the combo treatment significantly prolonged survival comparing to the control treatment and the STING-A treatment(Fig. 1E).

STING-A in combination with anti-PD-1 antibody enhances effector T cells infiltration and CD103 + dendritic cells in both target liver metastatic lesions and non-target metastatic lesions

To understand the mechanistic basis of the enhanced anti-tumor activity of STING-A in combination with a-PD-1, we performed flow cytometry analysis of the tumor infiltrating leucocytes(TILs) derived from the targeted liver metastasis in the same PDAC mouse model.

As described in the dosing schema in Fig. 1B, mice received the a-PD-1 on days 14 and 17, and the STING-A on days 14. On day 21, the mice were sacrificed and the implanted SubQ tumors as well as livers were harvested. TILs from the targeted liver metastatic lesion and the whole liver tissue excluding the targeted lesion were compared. Note that the whole liver tissue harvested on day 21 was diffusely infiltrated by non-target metastatic lesions. The results showed that the STING-A+a-PD-1 combo therapy significantly increased the infiltration of the CD8 + and CD8 + PD-1 + T cells, but not the CD4 + and CD4 + PD-1 + T cells in the target lesion comparing to treatment controls(Fig. 2A;Fig. S1C). Interestingly, a-PD-1, STING-A, or their combo all decreased the MHCII + CD11c + dendritic cells(DCs); however, comparing to the treatment control, a-PD-1 alone, or STING-A alone, the a-PD-1 and STING-A combo significantly increased the CD11b -CD103 + subtype of DCs(Fig. 2B), which are known to play a role in the cross-presentation of tumor antigens [START_REF] Joffre | Cross-presentation by dendritic cells[END_REF]. TILs from nontarget liver metastases showed that the combo treatment resulted in a significant increase in CD8 + T cells comparing not only to the treatment control, but also to a-PD-1 alone(Fig. 2C).

The infiltration of CD8 + PD-1 + T cells in the combo group was comparable with the control group and significantly lower than that in the STING-A alone group(Fig. S1D). Moreover, CD4 + T cells and CD4 + PD-1 + T cells were both significantly decreased in the combo treatment group compared to the STING-A treatment group, presumably due to the treatment effect of a-PD-1(Fig. 2C). These results suggest that, in non-target liver metastatic lesions, CD8 + and CD4 + T cells both trended in the favor of anti-tumor immune response following the a-PD-1 and STING-A combo treatment. In non-target liver metastases, MHCII + CD11c + DCs were similar among all treatment groups, suggesting that an enhanced antigen presentation was originated in locally targeted lesions. However, CD11b -CD103 + DCs were significantly elevated in the combo treatment group in non-target lesions(Fig. 2D). It is possible that CD103 + DCs trafficked from targeted lesions to non-target lesions. Taken together, these results suggest that both STING-A and a-PD-1 are required to activate local immune response in favor of anti-tumor response and that this immune response is extended to the non-target lesions in the vicinity of the target lesion.

STING-A in combination with anti-PD-1 antibody activate anti-tumor immunity via innate immune response signaling pathways

As the flow cytometry analysis has limitation in examining cytokine/chemokines and intracellular signals involved in the innate immune response, we examined the gene regulation of innate immune response following STING-A and/or a-PD-1 treatment by using the NanoString assays with the murine PanCancer Immune panel. We selected differentially expressed genes by comparing the STING-A and a-PD-1 combo treatment and treatment controls, STING-A alone or a-PD-1 alone with a 5% false discovery rate(Fig. S1E). We first examined the gene expression in the interferon(IFN) response pathways. As shown in Fig. 2E, the gene expression of IFN-(Ifng) was significantly increased in the combo treatment group and was also accompanied by significantly increased expression of Ifngr1, Ifnar2, Irf1, Irf4, Irf5, Irf7, Irf8, Stat1, Stat2, Stat4, Ifitm1, Ifih1, Ifit1, Ifit2, Ifit3, Ifi35, and Ifi44 compared to the control or single treatment groups. However, Irf3 was significantly decreased in the combo treatment group compared to the a-PD-1 treatment group(Fig. S1F). In addition, the tumor necrosis factor(TNF)-response pathway genes including Tnf(encoding TNF-α), Lta(encoding TNF-β), Nfkb1, Nfkb2, and Tank were also significantly upregulated in the combo treatment group compared to the control or single treatment groups(Fig. 2F). The innate immune response pathways genes including Tmem173(STING), Ddx58, Nlrp3, Nlrc5, Myd88, Clec4a2, Clec4n, Clec5a, Clec7a, Nod1, and Nod2 were significantly increased in the combo treatment group compared to the control or single treatment groups(Fig. 2G). However, as anticipated, the gene expression of Jak1 and Jak2 was similar among all treatment groups, suggesting Jak1/2 are not regulated at the RNA level(Fig. S1F).

We next investigated the expression of cytokines which are known to be involved in the development and differentiation of T and B lymphocytes(Fig. 3A-B;Fig. S2A-B). As shown in Fig. 3A, the expression of most genes in the interleukin(IL)-1 and IL-18 family except Il18 itself and Irak1 was significantly increased in the combo treatment group compared to most of other treatment groups. As demonstrated in Fig. 3B, the gene expression of most of the proinflammatory cytokine receptors including Il2ra, Il2rb, Il2rg, Il7r, Il12rb1, Il12rb2, l13ra1, Il13ra2, and Il15ra was significantly increased in the combo treatment group compared to most of other treatment groups. Interestingly, the cytokines themselves including Il2, Il7, Il12, and Il13, besides Il21 which was significantly upregulated, were upregulated in a non-statistically significant trend in the combo treatment group compared to the control or single treatment groups(Fig. S2A). These results suggest that pro-inflammatory pathways including those that mediate the inflammasome are activated broadly by the combination of the STING-A and a-PD-1.

As anticipated, we found that the majority of chemokine genes were upregulated in the combo treatment group compared to the a-PD-1 treatment group(Fig. S2C), likely due to the innate immune response induced by the STING-A. Therefore, we focused on those associated with T cell trafficking. Our results demonstrate a significantly increased expression of Ccl1, Ccl2, Ccl3, Ccl4, Ccl5, Ccl7, and Ccl8 in the combo treatment group compared to the vehicle control treatment group(Fig. 3C), suggesting that stimulation of innate immune response is anticipated to induce myeloid cell infiltration. C-X-C motif chemokine ligand(CXCL) 9, CXCL10, and CXCL11 are known to bind C-X-C motif chemokine receptor(CXCR) 3 on T cells and, in response to IFN signaling to recruit memory and activated effector T cells [START_REF] Tokunaga | CXCL9, CXCL10, CXCL11/CXCR3 axis for immune activation-a target for novel cancer therapy[END_REF].

We observed a significant enhancement of the expression of Cxcl9, Cxcl10, and Cxcl11 in the combo treatment group compared with the control or single treatment groups(Fig. 3C) although the expression of Cxcr3 was similar among different treatment groups(Fig. S1E).

Interestingly, comparing to the vehicle control treatment group, the administration of a-PD-1 showed a statistically non-significant trend of increase whereas STING-A showed a trend of decease in the expression of Ccl17, which encodes a T regulatory cell(Treg) chemokine. This finding is thus consistent with published studies showing that ICIs upregulate C-C motif chemokine ligand(CCL) 17 expression in tumors and increase the migration of Tregs into the TME of PDAC [START_REF] Mizukami | CCL17 and CCL22 chemokines within tumor microenvironment are related to accumulation of Foxp3+regulatory T cells in gastric cancer[END_REF][START_REF] Marshall | Tumors establish resistance to immunotherapy by regulating Treg recruitment via CCR4[END_REF]. Moreover, we here observed that the combo treatment led to a significantly decreased expression of Ccl17(Fig. 3C;Fig. S2D). It should be noted that the gene expression results from the NanoString assay may be influenced by an influx of immune cells that express the genes. Therefore, an increased expression of certain immune genes may represent an increased infiltration of the relevant immune subtypes. Taken together, these results suggest that STING-A may confer an antitumor effect by suppressing CCL17 expression or CCL17-expressing cells and thereby suppressing Treg migration into the TME.

STING-A in combination with anti-PD-1 antibody enhances T cell activation signals

Among those differentially expressed genes in the above NanoString analysis, we did further statistical analysis on genes related to the activation of effector T cells. As shown in Fig. S2E and as anticipated, the expression of T cell receptor/CD3 signaling pathway genes including Cd3d, Cd3e, Cd3g, Cd3z, Cd8a, and Lck in the combo treatment group was significantly increased [START_REF] Guy | Distinct TCR signaling pathways drive proliferation and cytokine production in T cells[END_REF][START_REF] Lipp | Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells[END_REF]. In addition, our results(Fig. 3D) revealed that Gzma, Gzmb, Gzmk, and Prf1 expression was significantly elevated in the combo treatment group comparing to most of other treatment groups, suggesting that the cytotoxic function of effector T cells are significantly enhanced. The gene expression of T cell co-stimulatory factors including Tnfrsf9(CD137), Tnfrsf4(OX40), and Icos were significantly increased in the combo treatment group when compared to any other group and including Cd27 when compared to the vehicle treatment group(Fig. 3E). However, the gene expression of co-inhibitory receptors including Pdcd1(PD-1), Lag3, Havcr2(TIM3), and Ctla4 and the expression of immune checkpoint activators such as Cd274(PD-L1) and Ido1 were significantly elevated in the combo treatment group compared to any other treatment group(Fig. 3F;Fig. S2F). These results suggested that T cell activation in the combo treatment group may also lead to the T cell exhaustion, in consistence with previously published studies [START_REF] Wherry | Molecular and cellular insights into T cell exhaustion[END_REF].

STING-A in combination with anti-PD-1 antibody enhances the infiltration and activation of effector T cells in the distant subcutaneous tumors

We next examined the immune response that mediates the abscopal effects in the distant tumors. As shown in Fig. 4A, we observed a significant increase in the infiltration of CD8 + and CD8 + PD-1 + T cells in the SubQ tumors from the combo treatment group, supporting the abscopal effect. Similar to the locally targeted lesions, CD4 + and CD4 + PD-1 + T cells in the SubQ tumors were not significantly changed among all treatment groups.

CD11b -CD103 + DCs in the SubQ tumors were also similar among all treatment groups, suggesting that the activation of antigen presenting cells occurred locally(Fig. 4B).

We then used the same NanoString assay to assess the T cell functional status. The results indicate that the expression of genes associated with the T-cell receptor CD3 complex exhibited a similarly significant increase in the SubQ tumors from the combo treatment group compared to other treatment groups as in the liver metastases(Fig. S3A). Similarly, genes related to the cytotoxic activities of effector T cells, including Gzma, Gzmb, Gzmk, and Prf1, demonstrated a significant increase in the combo treatment group compared to most of other treatment groups(Fig. 4C). In addition, the combo treatment group exhibited a significant increase in the expression of signals related to T cell activation including Cd27, Icos, Cd274(PD-L1), and Ido1 comparing to most of other treatment groups(Fig. 4D-E) and including Cd28, Cd80, Pdcd1(PD-1), Lag3, and Ctla4 comparing to the vehicle treatment group(Fig. S3B). Consistently, genes related to chemokines for effector T cell trafficking including Cxcl9, Cxcl10, and Cxcl11 exhibited a significant increase in their expression in the combo treatment group compared to most of other treatment groups(Fig. 4F). Ccl17 also exhibited a significant decrease in the combo treatment group compared to the vehicle treatment group. Interestingly, the expression of Ccl28, a T and B cell homing factor [START_REF] Mohan | CCL28 chemokine: an anchoring point bridging innate and adaptive immunity[END_REF],

showed a significant increase in the combo treatment group in SubQ tumors(Fig. 4F), but not the target metastatic lesions(Fig. S2C). The gene expression of cytokines and cytokine receptors that are relevant to the activated status of T cells was similarly increased as seen in the liver metastases, again except Il18(Fig. 4F), further supporting the abscopal effect.

In addition, the profiles of other cytokines and chemokines in the SubQ tumors were similar to those in the targeted liver metastases and showed a significant increase in proinflammatory immune responses in the IFN(Fig. 4G) and TNF pathways(Fig. 4H), but no significant changes or a decrease in Jak1 and Jak2(Fig. S3C), in the combo treatment group compared to other treatment groups. Nevertheless, expression of innate agonist receptors and adaptors in SubQ tumors appeared to be somewhat different from that in targeted liver metastases, showing an increase in Tlr8, Nlrc5, Nlrp3, Clec4n, Clec5a, Clec7a, and Nod1, but a decrease in Ticam1 and Mavs in the combo treatment group compared to other treatment groups(Fig. 4I). However, the profile of chemokines and chemokine receptors that function in the myeloid cell trafficking was similar between targeted liver metastases and distant SubQ tumors in the combo treatment group compared to other treatment groups(Fig. S3D).

Establish a mouse model for systemic administration of STING-A in combination with anti-PD-1 antibody induced both systemic and intratumoral immune responses

We next tested the IM administration, a systemic administrative route, of STING-A. To compare the anti-tumor efficacy of IM with IT injection of STING-A, we used the same liver metastasis model with implantation of SubQ tumors by following the same schema described in Fig. 1B. Firstly, systemic administration did not show any obvious toxicity including bleeding, unhealed wound, paralysis, or weight loss, etc. All the mice following the hemisplenectomy procedure were candidates for IM injection although we chose those feasible for IT injection for the purpose of comparison. We then found that mice in the combo(IM) group had a significantly prolonged survival when compared to other treatment groups(Fig. 5A). The TGI rate for SubQ tumors also exhibited a significant increase in the combo(IM) group(maximum TGI=58.86 ± 49.12%, p <0.05)(Fig. 5B). By combining the survival data in both IT and IM experiments, we found that the majority of mice died at around 3 to 4 weeks after tumor implantation whereas those who received both STING-A by IT or IM and a-PD-1 may live up to 6 weeks(Fig. S4A). Mice who received STING-A intramuscularly in combination with a-PD-1 reached the longest survival beyond 6 weeks although, likely due to the small sample size, there was no significant survival difference between the IM combo and the IT combo group.

To assess the systemic immune response induced by the IM injection of STING-A and/or a-PD-1, we first measured the cytokine response in the serum samples harvested 6 hours after the IM injection of STING-A. The results demonstrated that a number of cytokines especially those associated with inflammation had a significantly increased level in the sera of mice from the combo treatment group, including TNF-α, and IFN-γ(Fig. 5C). Interestingly, several T lymphocytes trafficking chemokines including CXCL9, and CXCL10 and type I cytokines including IL-2 [START_REF] Ross | Signaling and function of interleukin-2 in T lymphocytes[END_REF] and IL-12 [START_REF] Gately | Regulation of human cytolytic lymphocyte responses by interleukin-12[END_REF] were significantly increased in the sera from mice in the combo treatment group compared to other treatment groups, suggesting that IM injection of STING-A in combination with a-PD-1 is potentially able to induce an anti-tumor systemic immune response(Fig. 5D). In addition, some interleukins including IL-5 [START_REF] Molfino | Molecular and clinical rationale for therapeutic targeting of interleukin-5 and its receptor[END_REF], IL-6 [START_REF] Hirano | IL-6 in inflammation, autoimmunity and cancer[END_REF], and IL-10[32] that support lymphocyte growth and/or antibody production were also boosted in the IM combo group(Fig. S4B). The results also indicated the increased production of cytokines that participate in the recruitment of macrophages, neutrophils, and eosinophils, including CCL2, CCL3, CCL4, CCL5, CXCL1, granulocyte colony stimulating factor(G-CSF), and CCL11(Fig. S4C). These results suggested that the IM injection of STING-A is able to induce similar systemic immune responses as evidenced by the above described Nanostring analysis following the IT injection of STING-A.

Next, we assessed TILs by dissecting a single target liver metastatic lesion and a mixture of non-target liver metastases, respectively, in this model treated by IM injection of STING-A.

The single target liver lesion was pre-selected as it would be selected for the IT treatment, but without any IT treatment to be given(Fig. 5E-H;Fig. S4D-E). As shown in Fig. 5E-F, IM injection of STING-A alone significantly enhanced the infiltration of CD8 + and CD4 + T cells in both the pre-selected liver metastatic lesion and other liver metastases. The enhancement of the T cell infiltration was not as high in the IM combo group as the IM STING-A alone group. Nevertheless, CD103 + DCs showed a similar profile in the tumors with the IM injection of STING-A(Fig. 5G-H) compared to those shown above with the IT injection of STING-A, suggesting that systemic IM injection of STING-A is able to activate the desired antigen-presenting process in the liver metastases.

We then performed the NanoString analyses of liver metastases and SubQ tumors and compared them among treatment groups. As shown in the Fig. S5A, the high-throughput analysis of the NanoString results only revealed a smaller number of genes that were differentially expressed among the treatment groups than those differentially expressed among the treatment groups with the IT injection of STING-A(Fig. S1E). The results however supported a non-significant increasing trend in the expression of genes associated with the IFN response pathways in the liver metastases in the IM combo group(Fig. S5B).

Nevertheless, in the STING-A IM alone treatment group, the expression of T cell costimulatory molecules including Tnfrsf4(OX40), Cd27, Tnfrsf9(CD137), and Icos exhibited a trend(Fig. S5C) consistent with the results in the single STING-A IT treatment group(Fig. 3E).

However, the IM injection of STING-A combined with a-PD-1 did not significantly increase the expression of these co-stimulatory molecules, together with the above T cell infiltration results(Fig. 5E-F), suggesting a-PD-1 or a-PD-1 alone may not be an optimal immune checkpoint inhibitor treatment strategy in combination with STING-A. Nevertheless, we observed a strong trend of increased expression of genes related to the cytotoxic activities of effector T cells in the IM combo group including Gzma, Gzmb, Gzmk, and Prf1(Fig. S5D).

Chemokines especially those involved in effector T cell trafficking including Cxcl9, Cxcl10, and Cxcl11 exhibited similar trend as those genes associated with cytotoxic activities of effector T cells(Fig. S5E). In contrast, the increase in the T cell exhaustion and immune checkpoint signals and myeloid cell-recruiting cytokine/chemokine signals that were observed with the IT combo treatment were not observed in the IM combo group(Fig. 6A).

We also examined the immune response in the SubQ tumors in the experiment with the IM injection of STING-A. As shown in Fig. S6A, we observed a significant decrease in the infiltration ratio of CD4 + PD-1 + T cells in the CD4 + T cells in the SubQ tumors from the IM combo treatment group. A similar decreasing trend of CD8 + PD-1 + T cells was observed(Fig. S6A), demonstrating the treatment effect of a-PD-1. Next, we used the same NanoString assays to assess the T cell functional status within the SubQ tumors. Interestingly, unlike the results in the liver metastases, the expression of genes associated with the T-cell receptor CD3 complex exhibited a significant increase(Cd3z) or a strong increasing trend in the SubQ tumors from the combo treatment group compared to other treatment groups(Fig. S6B). T cell co-stimulatory molecules including Tnfrsf4(OX40), Tnfrsf9(CD137), Cd80, and Icos all exhibited an increasing trend(Fig. S6C). In addition, the IM combo treatment group exhibited a significantly increased expression of Il7r(Fig. S6D), which has been shown to play a critical role in the development of lymphocytes in the process known as V(D)J recombination [33]. These results suggest that there may be an intertumoral heterogeneity in the immune response to the systemic administration of STING-A. However, we found that the T cell exhaustion and immune checkpoint signals exhibited an enhanced expression in the IM combo treatment group compared to other treatment groups(Fig. 6B).

These results suggest that an enhanced T cell activation status and cytotoxic function in response to either IT or IM treatment of STING-A is associated with upregulation of T cell exhaustion signals and CTLA-4.

The overall increasing trend of those chemokines/chemokine receptors involved in the myeloid cell recruitment was less significant in both liver metastases(Fig. S5E) and SubQ tumors(Fig. S6D) than that in the IT combo group, suggesting that systemic delivery of STING-A does not lead to a strong induction of immunosuppressive signals. It is noteworthy to mention that Ccr5 expression in the SubQ tumors was significantly reduced by the treatment of a-PD-1 compared to the vehicle treatment group, but was significantly enhanced following the IM injection of STING-A in combination with a-PD-1(Fig. S6E). This result appears to be in consistent with the agonistic effect of C-C motif chemokine receptor(CCR) 5 expression previously reported in the PDAC models [START_REF] Wang | CCR2/CCR5 inhibitor permits the radiation-induced effector T cell infiltration in pancreatic adenocarcinoma[END_REF].

In addition to above differences between tumors from IM treated groups and IT treated groups, we noted that the expression of immunosuppressive myeloid cells associated-genes including Apoe, Trem2, Cxcr4, and Abcg1 was significantly increased in the SubQ tumors from the IM combo treatment group, but not the SubQ tumors from the IT combo treatment group, compared to the respective control group(Fig. 6C-F). Such a difference was not observed in the comparison between liver metastases from the IM combo treatment group and the IT combo treatment group(Fig. S6F-I). These results suggest that systemic administration of STING-A could still induce certain immunosuppressive signals that require additional targeted treatments.

STING-A in combination with immune checkpoint inhibitors significantly improved the

survival of genetically engineered KPC mice that develops invasive PDAC spontaneously.

Thus, above results suggest that systemic IM administration of STING-A led to less induction of immunosuppressive signals while maintaining a systemic and intratumoral immune response that favors antitumor response. These results also provide the mechanistic basis of the enhanced anti-tumor activity of the IM STING-A observed in the implanted tumor model(Fig. 5). We next validated this antitumor activity in the genetically engineered KPC mice that develops invasive PDAC spontaneously in a manner resembling human PDAC pathogenesis. As CTLA-4 remains to be one of immunosuppressive signals presented in the tumors treated by IM STING-A, we included anti-CTLA4 antibody to the immune checkpoint inhibitor regimen. KPC mice were subjected to weekly to twice weekly ultrasonic screening at age of 3 months and enrolled in the experiment once either the length, width, or height of the pancreatic tumor reached 2.00 mm to ensure eligible mice had equivalent tumor burdens. As shown in Fig. 7A, eligible KPC mice were randomized into the three treatment groups to receive a total of seven injections. Treatment toxicity and mouse survival were monitored for 3 months following the first treatment. No treatment related toxicity including local toxicity related to IM injection sites was observed. The STING-A monotherapy did not show any antitumor activity in a later experiment(manuscript in preparation). Dual checkpoint inhibitors failed to improve the overall survival of KPC mice when compared with control group; however, the co-administration of IM STING-A with dual checkpoint inhibitors significantly prolonged the survival of the KPC mice(Fig. 7B).

Systemic administration and intratumoral administration of an NLRP3 agonist in combination with anti-PD-1 antibody has a similar efficacy

In this study, we attempted to understand whether systemic administration can be applied to another innate immune agonist. We found that the combination of IT injection of NACHT, LRR, and PYD domains-containing protein 3(NLRP3) agonist and a-PD-1 had similar antitumor efficacy as the combination of IT injection of STING-A and a-PD-1 and prolonged the survival of liver metastasis mice implanted with SubQ tumors as compared to a-PD-1 alone, NLRP3 agonist alone, or vehicle control(Fig. S7A). Likely due to the small sample size, the combination of NLRP3 agonist and a-PD-1 failed to induce a significantly stronger tumor growth inhibition on either liver metastatic lesion or the SubQ tumors than single treatment groups(Fig. S7B-C). However, different routes of administration of NLRP3 agonist including IT, tail-vein injection, or intra-subcutaneous tumor injection resulted in no survival difference of the liver metastasis mice(Fig. S7D). Despite a small sample size, NanoString analysis showed that the expression of Gzmk(Fig. S7E), an effector T cell cytotoxicity-associated gene, was significantly increased in the NLRP3 agonist-treated tumor, but not in the STING-Atreated tumor(Fig. 3D), suggesting that further investigation of systemic administration of other innate immune agonists such as NLRP3 agonist is warranted.

Discussion

To our knowledge, this study is one of the few exploring the effects of the STING-A in combination with ICIs for the treatment of PDAC in a pancreatic liver metastasis murine model. We previously used this mouse model to support the application of STING-A as an adjuvant for the vaccine therapy. This mouse model resembles the spontaneous development of liver metastases in human PDACs and has been used in multiple prior studies for the preclinical development of rationale immunotherapy combinations [START_REF] Kim | Anti-pancreatic tumor efficacy of a Listeria-based, Annexin A2-targeting immunotherapy in combination with anti-PD-1 antibodies[END_REF][START_REF] Muth | CD137 agonist-based combination immunotherapy enhances activated, effector memory T cells and prolongs survival in pancreatic adenocarcinoma[END_REF][START_REF] Blair | Dual stromal targeting sensitizes pancreatic adenocarcinoma for anti-programmed cell death protein 1 therapy[END_REF]. The TME of the liver metastases in this model is similar to that of orthotopically implanted KPC tumors [START_REF] He | Preclinical mouse models for immunotherapeutic and non-immunotherapeutic drug development for pancreatic ductal adenocarcinoma[END_REF]. Our study demonstrated the antitumor efficacy of the combinational treatment with STING-A and PD-1 blockade in this liver metastasis model as well as novel evidence that demonstrated the survival benefit of the combination of a STING-A and ICIs in the genetically engineered, spontaneously formed KPC tumor model. In our model, STING-A in combination with a-PD-1 enhances effector T cells infiltration and CD103 + dendritic cells in both target liver metastatic lesions and non-target metastatic lesions as well as distant tumor lesions that are resembled by subQ tumors. More importantly, this study demonstrated that systemic administration of STING-A is equivalent to the intratumoral injection in both antitumor efficacy and immune response. Neither systemic nor intratumoral administration resulted in obvious toxicities; however, systemic administration was more feasible than intratumoral administration and also avoided any intragenic liver injury. Our study also supported the feasibility of administrating an NLRP3 agonist systemically and would supports a new paradigm of the clinical development of innate immune agonists by systemic administration.

Our results suggest that IM injection of STING-A is able to induce similar systemic immune responses as the IT injection of STING-A. Systemic IM injection of STING-A is also able to activate the desired antigen-presenting process in the liver metastases. Although the effector T cell responses appear to be slightly weaker in the IM injection of STING-A, the increase in the T cell exhaustion and immune checkpoint signals and myeloid cell-recruiting cytokine/chemokine signals that were observed with the IT combo treatment were not observed in the IM combo group.

Although this study did not demonstrate a significant difference in the treatment response between IM and IT injections of STING-A, we observed that mice who received IM injections of STING-A in combination with a-PD-1 survived longer than 6 weeks whereas none of the mice who received IT injection of STING-A in combination with a-PD-1 survived longer than 6 weeks. Therefore, it might be possible to see the survival benefit of the IM injection of STING-A if the sample size would potentially be larger; however, the sample size in each experiment was limited by the technical difficulty of IT injection. As it would be a challenge to breed a large number of the KPC transgenic mice for being randomized to multiple treatment groups, we decided to test the combination of a-PD-1 and anti-CTLA-4 antibody instead of two ICIs by itself in the experiments with the KPC transgenic mice. Our results suggest that an enhanced T cell activation status and cytotoxic function in response to either IT or IM treatment of STING-A is associated with CTLA-4 upregulation. Hence, the reason we included anti-CTLA-4 antibody to a-PD-1 in the experiment with the KPC transgenic mice. Future studies could compare the combination of a-PD-1 and anti-CTLA-4 antibody with either a-PD-1 alone or anti-CTLA-4 antibody alone.

This study has a few limitations, first being the smaller sample size. As discussed above, the sample size is limited by the technical complexity of IT injection and breeding KPC transgenic mice. However, the sample size in the current study is, to our knowledge the largest one in testing the intratumor injection of the tumors in internal organs. It is also one of the largest using the KPC transgenic mice as the preclinical model for the efficacy testing.

However, the current sample size allowed this study to repeat most of the experiments.

Second, this study did not examine the effect of anti-CTL4-4 antibody separately from the treatment groups testing the combination of a-PD-1 and anti-CTLA-4 antibody. Third, chemotherapy, as a current standard of care treatment, was not included in the experiments in this study. We were concerned that chemotherapy would complicate our comparison of the antitumor efficacy and immune response between the IM and IT injection of STING-A. We are studying the combination of chemotherapy and STING-A in an independent study.

To test the feasibility of systemic administration of other innate immune agonists, we examined the NLRP3 agonist in this study. Although it may have been limited by the small sample size, this study demonstrated that there were no significant differences in anti-tumor efficacy between intratumoral and systemic administration of NLRP3 agonist. This study has not performed an in-depth investigation on the NLRP3 agonist. However, with limited data, this study suggests that further investigation of systemic administration of other innate immune agonists such as NLRP3 agonist is warranted. The results in this study have thus supported the phase 1 trial that evaluated BMS-986301 intratumoral or intravenous injection

Figures and

Figure 1 .

 1 Figure 1. Intratumoral injection of the STING agonist in combination with anti-PD-1 antibody enhance local anti-tumor efficacy with abscopal effects. (A) Ultrasonographic measurement for the target liver metastatic lesion before and after intratumoral injection of the STING agonist. Arrow indicates the injection needle path. (B) Treatment schema. Mice that met inclusion criteria were randomly assigned to each group on day 12 and monitored twice a week until survival endpoints were met. (C) Tumor Growth Inhibition (TGI) of the injected liver metastatic lesion during the treatment period. (D) Tumor Growth Inhibition of the remote subcutaneous (SubQ) tumor during the treatment period. Dashed line at -50% indicates statistically significant TGI. (E) Kaplan-Meier survival curves compare different treatment groups. NC, vehicle/isotype antibody control; STING A, STING agonist; a-PD-1 Ab, anti-PD-1 antibody; Combo, STING A+a-PD-1 Ab; IT, intratumoral; IM, intramuscular; IP, intra-peritoneal. Data shown as mean ± SD; comparison by unpaired t test in C and D, and by Log-rank test in E; *p < 0.05; **p < 0.01; NS, not significant.

Figure 2 .

 2 Figure 2. STING agonist in combination with anti-PD-1 antibody enhances effector T cells and CD103 + dendritic cell (DC) infiltration and activates anti-tumor immunity via innate immune response signaling pathways. (A) Percentages of the CD8 + and CD8 + PD-1 + T cells among CD45 + leucocytes in the target liver metastatic lesion. (B) Percentages of the MHCII + CD11c + DC among CD45 + leucocytes and the percent of CD11b -CD103 + DC subtype in the target liver metastatic lesion. (C) Percentages of the CD8 + , CD4 + , and CD4 + PD-1 + T cells among CD45 + leucocytes in the non-target liver metastases. (D) Percentages of MHCII + CD11c + DC among CD45 + leucocytes and the percent of CD11b -CD103 + DC subtype in the non-target liver metastases. (E) Expression of genes in the IFN-response pathways in the target liver metastatic lesions from different treatment groups. (F) Expression of genes in the TNF-response pathways in the target liver metastatic lesions from different treatment groups. (G) Expression of genes in the innate immune response pathways in the target liver metastatic lesions from different treatment groups. Data are shown as the mean ± SD; comparison by unpaired t test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.Remaining comparisons are non-significant.

Figure 3 .

 3 Figure 3. Differentially expressed genes in the target liver metastatic lesions between treatment groups. Expression of genes, as indicated, in the gene families of cytokines (A-B), chemokines (C), the activation of effector T cells (D), the T cell co-stimulatory factors (E), and the immune checkpoint activators (F). Data shown as mean ± SD; comparison by unpaired t test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Remaining comparisons are non-significant.

Figure 4 .

 4 Figure 4. STING agonist in combination with anti-PD-1 antibody enhances the infiltration and activation of effector T cells in the distant subcutaneous tumors. (A) Percentages of immune cells in the remote SubQ tumors, such as the CD8 + , CD8 + PD-1 + , CD4 + , and CD4 + PD-1 + T cells among CD45 + leucocytes, the CD8 + PD-1 + among CD8 + T cells, and the CD4 + PD-1 + T cells among CD4 + T cells, respectively. (B) Percentages of the MHCII + CD11c + DC, CD11b -CD103 + and CD11b + CD103 -DC subtypes DC among CD45 + leucocytes, respectively, in the distant SubQ tumors. Differentially expressed genes in the distant SubQ tumors from different treatment groups, including those in the gene families of cytotoxic T cell function (C), T cell activation (D), and the immune checkpoint activators (E). (F) Heatmap of the interleukin family genes and chemokine genes that were significantly increased in the Combo group comparing to the vehicle control treatment group. (G) Expression of genes in the IFN-response pathways in SubQ tumors from different treatment groups. (H) Expression of genes in the TNF-response pathways in SubQ tumors from different treatment groups. (I) Expression of genes in the innate immune response pathways in SubQ tumors from different treatment groups. Data shown as mean ± SD; comparison by unpaired t test; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Remaining comparisons are non-significant.

Figure 6 .

 6 Figure 6. Comparison of expressions of differentially expressed genes in tumors from systemically treated mice. Expression of genes of the checkpoint gene family in pre-selected, target single liver metastatic lesions (A) and distant subcutaneous tumors (B) from the intramuscularly treated mice. Expression of Apoe (C), Trem2 (D), Cxcr4 (E), and Abcg1 (F) in the distant subcutaneous tumors from both intratumoral and intramuscularly treated mice. Data shown as mean ± SD; comparison by unpaired t test; *p < 0.05; **p < 0.01; ***p < 0.001. Remaining comparisons are non-significant.

Figure 7 .

 7 Figure 7. STING agonist in combination with immune checkpoint inhibitors significantly improved the survival of genetically engineered KPC mice. (A) Treat schema. (B) Kaplan-Meier survival curves compare overall survival between different treatment groups. NC, vehicle/isotype antibody control; STING A, STING agonist; a-PD-1 Ab, anti-PD-1 antibody; a-CTLA-4 Ab, anti-CTLA-4 antibody; IM, intramuscular; IP, intra-peritoneal. Data shown as mean ± SD; comparison by Log-rank test; *p < 0.05; **p < 0.01; NS, not significant.

Acknowledgments

We would like to acknowledge the important insight provided by Jie Fang, Gary Schieven, and Jordan Blum from the Bristol-Myers Squibb group. This work was done at the Johns Hopkins University. 759 760 786 787

Funding

This study was supported by a Bristol-Myers Squibb II-ON grant (L. Zheng). LZ is supported by an NIH Grant R01 CA169702, an NIH Grant R01 CA197296, an NIH Grant P01 CA247886, an NIH SPORE Grant P50 CA062924, and an NIH Cancer Center Support Grant P30 CA006973. KL is supported by a National Natural Science Foundation of China 82303740, a Key Research and Development Project of Science and Technology Department of Sichuan Province 2023YFS0167, a China Postdoctoral Science Foundation 2023T160451, and a West China Hospital Postdoctoral Science Foundation 2023HXBH053.

Availability of data and materials

All data needed to evaluate the conclusions in the paper are present in the paper and the Supplementary Materials. Any further information required to support our data will be supplied upon request.

as monotherapy or in combination with nivolumab(PD-1 blockade) and ipilimumab in patients with advanced solid cancers(NCT03956680).

Abbreviations

Declarations

Ethics approval and consent to participate

All studies and maintenance of mice were conducted in accordance with the approval of the Institutional Animal Care and Use Committee (IACUC) guidelines of Johns Hopkins School of Medicine (Animal Protocol: MO22M59).

Consent for publication

Not applicable.

Competing interests

L.Z. receives grant support from Bristol-Meyer Squibb, Merck, AstraZeneca, iTeos, Amgen, NovaRock, Inxmed, Halozyme and Abmeta. L.Z. is a paid consultant/Advisory Board Member at Biosion, Alphamab, NovaRock, Ambrx, Akrevia/Xilio, QED, Novagenesis, Snow Lake Capitals, Amberstone, Pfizer, Tavotek, and Mingruizhiyao. L.Z. holds shares at Alphamab, Amberstone, Mingruizhiyao, and Cellaration.

Authors' contributions

Keyu Li and Junke Wang contributed equally to this project. Concept was conceived by L.Z.

The strategy and the overall study were designed by K.L. and L.Z. Experiments were conducted by K.L., Junke W., N.N., N.J., A.B., Jiangxin W., M.H., J.Y., D.T., B.E., C.Y., and M. W. Data were collected by K.L. and Junke W. Formal analysis was conducted by K.L., Junke W, and Y.X. Genetically modified mice were generated by M.H, V.F., and M.L.

Original draft manuscript was written by K.L. Manuscript was reviewed and revised was by A.O., B.E., M.W., and L.Z. Supervision was made by X.L., Y.W., and L.Z. The project administrator is L.Z.