Decoding The Stem Cells-Immune Cell Dialogues For Cancer Immunotherapy.

Abstract Adult stem cells (SCs) reside in various body sites where they govern tissue homeostasis and orchestrate wound repair. As a group of long-lived and indispensable cells, they inevitably face the challenge of experiencing repeated bouts of inflammation initiated by tissue injury, infection, and noxious agents. Especially during injury repair, tissue SCs must be mobilized to exit their immune privileged niche, and enter a highly inflammatory environment in the center of the wound bed to regenerate the tissue. In order to successfully repair the damage, it is essential for SCs to be equipped with extraordinary abilities to adapt to immune responses. Recently, a subset of TGF-β-reactive tumor-initiating cells were found at the tumor-stroma interface near blood vessels of the squamous cell carcinomas (SCC), including the SCCs in skin or head & neck. These cells appear to be enriched with adult stem cells features, including strong tumor initiating capacity, and expression of typical SC surface markers (e.g. CD34, Integrin a6) or SC transcription factors (e.g. Sox2, Sox9, Klf5). More importantly, these cells can serve as the major source of malignant in skin squamous cell carcinoma. To drive cancer progression, it is also essential for these stem cell-like tumor-initiating cells to develop unique capacity to evade the anti-tumor immunity. However, so far, it is still unclear whether the adult stem cells are endowed with special immune evasive capacity and whether tumor-initiating stem cells can hijack this activity. This abstract is describing a series of our recent work, including unpublished data that are stimulated by our central vision to simultaneously examine and compare the stem cell-immune cell crosstalk in both wound healing of normal tissue and cancer regeneration after immunotherapy treatment. These studies have led to uncovering a special dialogue between skin stem cells and regulatory T cells which can be targeted as novel cancer immunotherapy. In order to explore immune resistance mechanisms that are unique to tumor-initiating stem cells, we designed a novel mouse model for cutaneous SCC that could be effectively challenged with adoptive T cell transfer (ACT)-based immunotherapy. This mouse harbors two transgenes, oncogenic HRASG12V and Ovabumin (OVA), both of which are controlled by doxycycline responsive element. We used a powerful in utero gene delivery method to introduce at low titer a lentiviral (LV) construct which we engineered to encode a Doxy-regulated transactivator rtTA. When injected into the amniotic fluid of E9.5 embryos, the low titer LV becomes stably and selectively integrated into a few skin epithelial cells. This approach allows us to couple the OVA and HRASG12Vexpression in only the few clonal skin patches that were transduced with the LV. With this novel system, we then activated OVA-targeting cytotoxic T cells in vitro, and use them to attack the spontaneous HRASG12V/OVA+ SCC tumors that develop on these mice. Importantly, the tumors cannot easily silence OVA expression because OVA is activated by the same elements driving oncogenic RAS expression, therefore, maintaining strong antigen presentation. Upon ACT treatment, we observed extensive OT1 T cell infiltration, and the OVA+ SCC tumors rapidly shrunk by 80-90% within four weeks. However, 70% of treated tumor eventually relapsed after few months. Remarkably, although the bulk of the tumor cells were efficiently cleared by the injected OT1 CTLs, some tumor cells always survived to give rise to the relapsed tumors. When we FACS-purified these ACT-surviving tumor cells and subject them to singe cell RNA-seq, we found majority of these ACT survivors were enriched for stem cell-associated genes, such as CD34, CD44, or Sox9, as well as a TGFβ responsive signature. We then designed a lineage-tracing experiment, and used a TGFβ reporter to track the fate of these TGFβ-responding tumor initiating stem cells after ACT treatment. We provided compelling evidence that a subset of TGFβ-responding skin cancer stem cells, selectively survive the ACT treatment and by lineage tracing, are responsible for the relapsed tumor that grows back following immunotherapy. This finding further strengthened our speculation that SCs must be endowed with unique capabilities to adapt to inflammation. To determine which self-protective programs are also activated by normal SCs after exposure to inflammation and after SCs exit their protective niche, we first established a partial-thickness removal cutaneous wounding mouse model. In this model, only the epidermis of the mouse skin is mechanically removed, forcing the hair follicle stem cells (HFSCs) in the dermis to leave the hair follicle, and enter a highly inflammatory environment in the wound bed to regenerate the lost epidermis. The HFSCs were genetically labeled the with red fluorescent protein in Sox9CreER;Rosa26loxp-STOP-loxp-tdTomatomice, so that we can track and isolate the migratory HFSCs from the wound bed and subject these cells to Assay for Transposase-Accessible Chromatin using sequencing (ATACseq) to determine which SC chromatin domains become accessible and activated. Surprisingly, ATAC-seq revealed strong activation of class II MHC and CD80, the cell-surface immune modulatory molecules canonically found on antigen-presenting immune cells. Activation of MHCII or CD80 was confirmed, as determined by flow cytometry, on non-immune epithelial populations, specifically on the subset of HFSCs that have migrated out of the hair follicles after wounding that acquired Integrin a5. To test the functional significance of these immune modulatory proteins for epithelial SCs during wounding, CD80 was targeted in a mouse model. Using both Cd80null mice reconstituted with wild type bone marrow, or employing the in utero lentiviral delivery system to deliver a Cd80small guide RNA to the epidermal epithelial progenitor layer of E9.5 K14Cre;Rosa26-Cas9-GFP Loxp-Stop-Loxpmice,Cd80was conditionally knocked out (cKO) in the basal epithelial layer where HFSCs are located. After wounding, Cd80cKO mice displayed significantly delayed re-epithelization and defective SC differentiation compared to control mice. Interestingly, when we profiled the immune composition of Cd80cKO wound, compared to the control wound, we observed significantly reduced regulatory T cells (Treg), resulting drastic accumulation of neutrophils in the wounds of Cd80cKO mice. Surprisingly, we further revealed that the tumor-initiating stem cells that have survived from the ACT treatment also acquired the immune modulatory factors that are activated in migratory HFSCs during wound healing to regulate Tregs, such as CD80. Consistent with the potential involvement of SC-Treg dialogues in driving tumor relapse from immunotherapy treatment, we observed a significant influx of Tregs in relapsed tumors. Additionally, abolishing these factors, such as CD80 significantly reduce the Treg infiltration into the skin tumors. Based on these observations, we can conclude that, during development of resistance and recovery from immunotherapy treatment, tumor-initiating cells acquire SC-specific immune modulatory program to dampen down the anti-tumor immunity and successfully drive tumor relapse. Cancer and wounds indeed share many characteristics, and cancer has been considered as “a wound that never heals”. Prior interpretations of this theory mainly pointed to the observations that similar molecular pathways drive stem cell proliferation, differentiation and stress responses in both wound and cancer. The intimate dialogue between stem cells and surrounding immune cells has long been overlooked. In this study, we have unveiled that the stem cell-immune cell crosstalk actually dictates the outcomes of successful tissue regeneration or tumor growth. These discoveries will provide a new revenue to leverage and target these vital crosstalk as novel cancer immunotherapy. Citation Format: Yuxuan Phoenix Miao, Cynthia Truong, Elaine Fuchs. Decoding the stem cells-immune cell dialogues for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG13.