CAFs contribute to Immunosuppression and Therapy Resistance
The tumor microenvironment (TME) limits the accumulation, distribution, and function of drugs and immune cells. This is mainly due to compressed vessels resulting from solid stress generated by proliferating cancer cells, CAFs, and the dense ECM secreted by CAFs [1,2]. The thick fibrotic tissue plays a key role in mediating tumor immune exclusion. CAFs also mediate immunosuppression through their immunomodulatory secretomes. This, in turn, affects immunotherapy outcomes necessitating new TME-modulating strategies to overcome therapy resistance.
CAF mediated immunosuppression
The tumor is immunosuppressed in the following cases:
1. The immune cells are absent within or surrounding the tumor (Immune desert phenotype)
2. Immune cells are restricted to the tumor periphery but not allowed inside (Immune restricted phenotype)
3. The tumor promoting immune phenotypes are abundant in the tumor (Immune inflamed phenotype)
The phenotypes 2 and 3 are mediated by CAFs and ECM [3].
Immune restricted phenotype: The immune cells are excluded from the tumor due to faulty vasculature and stroma in the periphery which is characterized by a high density of CAFs, increased expression of TGFβ, and excessive ECM.
· CAFs in the desmoplastic region of the tumors secrete TGFβ and stromal cell derived factor 1α, which prevent cytotoxic T lymphocytes (CTLs) from migrating to cancer cells, which is associated with a poor response to checkpoint immunotherapy (ICI) [4].
· Biopsy samples of lung metastases obtained from women with breast cancer also typically have a pattern of fibrosis that excludes T cells [5].
· CAFs have immunosuppressive subsets and are also able to produce and maintain the dense desmoplasia that blocks CTL migration [6].
o For example, Grout et al. identified two CAF populations associated with T cell exclusion: (i) MYH11+ αSMA+ CAF, which were present in early-stage tumors and formed a single-cell layer lining cancer aggregates, and (ii) FAP+ αSMA+ CAF, which appeared in more advanced tumors and organized in patches within the stroma or in multiple layers around tumor nests. Both CAF populations had a dense and aligned fiber deposition compared to T cell permissive CAFs. However, they produced distinct matrix molecules: collagen IV (MYH11+ αSMA+ CAF) and collagen XI/XII (FAP+ αSMA+ CAF) [7].
Immune inflamed phenotype: Certain CAFs recruit immune suppressive cells. CAFs are strongly immunomodulatory and influence the infiltration, phenotype of immune cells, their spatial location within tumor. They influence immune cell infiltration either directly — via secreted cytokines and chemokines and cell surface proteins — or indirectly — through deposition of various ECM components and remodeling of matrix on which immune cells depend for localization, migration, and intercellular interaction [8].
· Many studies have shown that CAFs produce immunomodulatory cytokines, such as IL-10, TGFβ, TNF, IFNγ, CCL2 and IL-6, and help to recruit immunosuppressive cells and/or polarize macrophages, T lymphocytes and natural killer cells [9–11].
o CAFs can secrete a number of factors known to influence both recruitment and activation state of myeloid cells including many chemokine ligands (CXCLs), IL-1β, IL-6, IL-8, IL-10, leukemia inhibitory factor (LIF), VEGF, TGF-β, prostaglandin (PG) E2 (PGE2), tumor necrosis factor (TNF), or nitric oxide (NO) [12].
o CAF-derived IL-6 drives STAT3 activation in myeloid cells, driving them to more immunosuppressive cell types such as regulatory dendritic cells [13].
o CAFs secrete IL-6 leading to an increased ratio of FoxP3+ T cells over CD8+ T cells, which was also associated with poor clinical outcome [14].
o TGFβ also induces the development of a Treg phenotype in naive CD4+ T cell precursors [15].
o Fibroblast IL-6 drives differentiation of other regulatory T cells such as interleukin-17 (IL-17)-producing T helper (TH17) cells. IL-17 released from TH17 cells can drive production of angiogenic factors from fibroblasts and cancer cells, thus fueling tumor growth [16].
· Clinical data shows correlation between stromal markers and immunosuppressive cell types such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) [17], which are correlated to immunotherapy response [18]. CAF markers reportedly correlate with immunosuppression of T cells in the TME.
Stromal strategies to overcome immunotherapy resistance
· “Normalization” of the tumor extravascular compartment by reprogramming cancer-associated fibroblasts (CAFs) to produce a less dense extracellular matrix (ECM) can be applied alone or in combination with immune checkpoints to normalize the entire TME and delivery of macromolecules, immune cells, and drugs. Improper use of these strategies, however, can lead to CAF and/or ECM depletion, which might accelerate tumor progression and metastasis [18]. This is one approach of converting immunosuppressed TME to immunostimulatory TME through drugs that normalize TME [19]. For example, Sherman et al. suppressed pancreatitis and improved pancreatic therapy by normalizing the TME through Vitamin D receptor-mediated stromal reprogramming [20].
· Stromal elements like ECM can also inhibit therapies and targeting ECM molecules could potentiate them. For example, collagenase-based therapies to inhibit collagen from limiting the diffusion of drugs and immunotherapies can be combined to improve therapy efficacy [21,22]. Also, MMP-1 and MMP-8 mediated depletion of tumor sulfated glycosaminoglycans or GAGs increases the distribution and efficacy of oncolytic viral vector therapy [23].
· Fibroblasts within the TME can express PD-L1/2 themselves and increase expression in tumor cells [24]. Even after CAR-T or presence of checkpoint inhibitors, the CAF comprising stroma acts as a barrier to T cells [25]. For example, MYH11+ αSMA+ CAFs and FAP+ αSMA+ CAFs can be targeted to increase immunotherapy efficacy in patients bearing T cell-excluded tumors.
· Drugs that interfere with TGF signaling have potential to improve immunotherapy . One promising strategy is to repurpose TGFβ inhibiting drugs like metformin, pirfenidone and tranilast in cancer. These are called mechanotherapeutics [26]. In mouse models of metastatic cancer, TGFβ promotes immune evasion, while inhibition of TGFβ signaling produces a potent T cell response [27] and potentiates the efficacy of ICI [28]. Inhibition of TGFβ signaling upregulates the expression of adhesion molecules on tumor blood vessels that are used by lymphocytes to extravasate [29]. Expression of TGF-β-associated ECM genes in CAFs is one of the strongest predictors of immunotherapeutic failure [30], and co-administration of anti-TGF-β antibodies and anti-PD-L1 antibodies significantly improved success of immunotherapy in preclinical models [17].
· CAF expression of JAM2, OX40L, and PD-L2, significantly increases the duration of contact time between T cells and CAFs. And, CAF expression of B7H3, CD73, and DPP4 play a more direct role in allowing CAFs to drive expansion of CD25+FOXP3+ T cell numbers [31]. Potential immunotherapies are developing around each target with success in early preclinical models [32].
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