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Cutaneous malignant melanoma is amongst the most aggressive cancer types, which typifies the paradox of being simultaneously highly antigenic and highly immunoevasive [1, 2]. However, various anti-melanoma immunotherapies have shown limited clinical success e.g. IL2, IFN-α2a or dendritic cells (DCs)-based vaccines [1–3]. Subsequent research showed that tumor-induced immunosuppression caused failure of above mentioned immunotherapies and thus overcoming this was proposed to be the key to success. This assumption proved correct, as evident by the impressive anti-melanoma clinical responses achieved with anti-CTLA4/PD-1/PD-L1 antibodies (i.e. immune-checkpoint inhibitors or ICIs) [2, 3]. However, despite this success, there remains a sizeable subset of melanoma patients that don't respond to ICI-therapies . While efforts are underway to dig out new immunological therapeutic targets yet recent research suggests that cancer cell-autonomous events can also play an important role in resistance to immunotherapies. For instance, recently melanoma cells were reported to mount resistance against anti-CTLA4 immunotherapy by up-regulating surface PD-L1 and thereby causing T cell exhaustion (Figure (Figure1)1) . Similarly, melanoma cell-autonomous WNT/β-catenin signaling was found to cause T cell exclusion from tumor microenvironment thereby creating ICI-therapy resistance (Figure (Figure1)1) .
The above research on cancer cell-autonomous resistance has largely focused on ICI-therapies. However, it is also widely recognized that a durable “resetting” of the tumor microenvironment towards immune-susceptibility requires effective treatment with cytotoxic drugs that activate anticancer immunity [4–6]. We believe that the highest degree of such “reset” is achievable by inducers of immunogenic cell death (ICD) [5, 6]. ICD is a cell death routine triggered by a limited set of assorted anticancer therapies (anthracyclines, radiotherapy, photodynamic therapy/PDT, oncolytic viruses), that is accompanied by danger signaling-driven, spatiotemporally defined, surface exposure or secretion/release of damage-associated molecular patterns (DAMPs) like surface-calreticulin (ecto-CRT, a quintessential ‘eat me’ signal that mediates immunogenicity) [4–6]. These DAMPs than interact with their cognate receptors, and help in instigating a potent anti-tumor immune response that helps eradicate residual cancer cells [4, 5]. ICD has shown significant preclinical promise in a number of experimental models and some clinical promise in patients of lung cancer, breast cancer, ovarian cancer and lymphoma [4–6].
Remarkably, melanoma, which shows palpable pre-clinical susceptibility to ICD, has usually failed to show clinical responsiveness to single-agent ICD inducers like, anthracyclines/doxorubicin . These paradoxical results have driven some of our recent research endeavors aimed at studying the ICD-melanoma link. We have found that melanoma cell-autonomous resistance to ICD can operate on two major levels i.e. macroautophagy activity [6, 8] (Figure (Figure1)1) and general capability of surface-exposing CRT [1, 4] (Figure (Figure1),1), as further discussed below.
Using a bona fide ICD inducer, Hypericin-based PDT (Hyp-PDT) , we found that human melanoma cell-associated macroautophagy suppresses exposure of ecto-CRT (possibly by reducing oxidative-ER stress), which in turn reduces maturation of the interacting DCs, DC-derived IL6 production and proliferation of IFN-γ producing CD4+/CD8+ T cells (Figure (Figure1)1) . Thus, these results unraveled a role for ROS-induced autophagy in weakening the functional interaction between dying melanoma cells and immune cells . These results were recently, partially, extended to BRAFV600E inhibitor-resistant melanoma cells where autophagy was shown to suppress exposure of ecto-CRT and ecto-HSP90  (Figure (Figure1).1). Nevertheless, it remains to be seen whether this activity of cell-associated autophagy also weakens the interactions between immune cells and BRAFV600E inhibitor-resistant melanoma cells .
In a recent report we demonstrated the existence of a broad ICD-resistance mechanism using an AY27 rat bladder cancer model and two bona fide ICD inducers (mitoxantrone and Hyp-PDT) . This ICD-resistant phenotype stemmed from low endogenous CRT protein levels in cancer cells (i.e. CRTlow-phenotype) which resulted in defective ecto-CRT levels (Figure (Figure1),1), which further caused severely reduced phagocytic clearance of treated cancer cells, which ultimately lead to the failure of tumor-rejecting immunity . Interestingly, we found that a subset of cancer patients of various cancer-types tend to exhibit CALRlow or CRTlow-tumors . Moreover, we observed that tumoral CALRhigh-phenotype was predictive of positive clinical responses to therapy with ICD inducers like radiotherapy or paclitaxel in non-small cell lung or ovarian cancer patients, respectively (but not non-ICD inducer like topotecan in ovarian cancer) . Additionally, tumoral CALR levels positively correlated with the levels of genes relevant for phagosome maturation or processing in only the clinical ICD set-up  (Figure (Figure1).1). Importantly, we found that a subset of melanoma patients also had the tendency to show CRTlow-tumors thereby hinting at the possible existence of above resistance mechanism in melanoma . Possibility of such resistance mechanism in melanoma is of high implication since our research has found ecto-CRT to be crucial for immunogenicity of dying melanoma cells . More specifically, we have shown that a well-established anti-melanoma chemotherapeutic, melphalan fails to induce sufficiently high immunogenicity in vivo, because it is not able to induce the relevant threshold levels of reactive oxygen species (ROS)-based ER stress required for ecto-CRT induction (Figure (Figure1)1) . Ecto-CRT on melphalan-treated melanoma cells was “restored” when the low ROS-based ER stress was increased by combining with the ER stressor, thapsigargin . Importantly, we also observed that dying melanoma cells were largely reliant on ecto-CRT for immunogenicity since ecto-HSP90, which was emphatically exposed by melphalan-treatment failed to mediate immunogenicity . In near future, it would be interesting to find whether overall CRT or CALR levels in melanoma are predictive of clinical responses to ICD or immunotherapy and/or regulate overall levels/spatial distribution of CD8+ T cell-infiltrates.
In conclusion, there clearly exist melanoma cell-autonomous mechanisms that disrupt responses to immunotherapy with ICI-drugs or ICD-inducers. However, a future exome-sequencing or deep-sequencing study utilizing melanoma patient samples is required to characterize melanoma genotypes that associate with poor T cell infiltration and/or clinical responses to anti-melanoma therapeutics.
CONFLICT OF INTEREST
No potential conflicts of interest were disclosed.