The tumor immune landscape: new insight into the spatial relationships and complex phenotypes of the immune cells during tumor development and response to therapy

The investigation of tumor microenvironment (TME) has become crucial to find predictive and prognostic markers that might contribute to the appropriate treatment choice. Here, combining gene expression profiling (GEP) and quantitative multiplex immunofluorescence (mIF), we describe the TME of Human papilloma virus (HPV)-driven and non-HPV-driven oropharyngeal squamous cell carcinoma (OPSCC) and of carcinoma in situ of the bladder (CIS) treated with hyaluronic acid (HA)-paclitaxel (PTX) conjugate. The analysis of the TME of 39 treatment-naïve OPSCC and the corresponding lymph node metastases unveiled a stronger activation of immune signaling pathways and a higher expression of genes related to total tumor-infiltrating lymphocytes, CD8 T cells, cytotoxic cells and exhausted CD8 cells in HPV-driven patients in comparison with non-HPV-driven counterparts. CD8+ T cells appeared closer to tumor cells, CD163+ macrophages and FoxP3+ cells in HPV-driven primary tumors and in corresponding metastases. In HPV-driven lesions, PD-L1 expression was more pronounced and PD-L1+ tumor cells and macrophages were closer to PD-1+ cytotoxic T lymphocytes. A positive correlation was observed between DFS and higher levels of activating immune signatures and T cell responses, higher density of PD-1+ T cells and their closer proximity to tumor cells or PD-L1+ macrophages. HPV-driven patients with higher infiltration of T cells and macrophages had a longer DFS, while CD163+ macrophages had a negative role in prognosis of non-HPV-driven patients. These results highlight the possibility to employ immune checkpoint inhibitors (ICI) in treatment-naïve patients with heavily infiltrated OPSCC, and to combine ICI with tumor-specific T cell response inducers or tumor-associated macrophages (TAM) modulators for the ‘cold’ counterparts. HA-PTX conjugate represents a potential alternative to standard approaches for the treatment of bladder CIS. Specimens deriving from the 20 patients with BCG-unresponsive CIS of the bladder treated with HA-PTX intravesical instillation for 15 months in the NCT04798703 trial were analyzed. Non-responders (NR) patients showed more intra-tumoral CTL density, but also more interactions between CD8+ T cells and TAMs or FoxP3+ cells. Macrophages tumor infiltration was associated with a shorter DFS. Bladder CIS cells expressed different CD44 isoforms. CD44v6 expression was higher in NR patients, and was associated with a shorter DFS. Moreover, CD44v6 expression was positively correlated with intra-tumoral macrophage density, and the combination of the assessment of CD44v6 expression and macrophage density represented a stronger predictive biomarker for patient response to HA-PTX. Thus, our results suggest an interplay between CD44v6 and macrophages that could play a role in HA-PTX response, suggesting to combine HA-PTX with treatments targeting the macrophage population.