https://www.nature.com/articles/s41587-022-01379-y

Although intended as a proof of concept that analyses of local nanodose drug responses can effectively guide systemic treatment strategies, we have already identified specific therapeutic combinations that warrant clinical consideration, including palbociclib/anti-CSF1R, venetoclax/anti-CD40, panobinostat/anti-PD-1 and lower-dose panobinostat/venetoclax/anti-CD40. Clinical work already underway suggests the feasibility of such evaluations. CDK4/6 inhibitors, including palbociclib, are FDA approved and considered as standard of care for patients with metastatic breast cancer^3,60. A smaller phase 1b study measured the safety and preliminary efficacy of venetoclax in patients with ER⁺BCL2⁺ breast cancer to be similar to other the ‘modern-day’ therapies⁶¹. Although CDK4/6 inhibitors can induce anti-tumor immunity in breast cancer⁶², in part through epigenetic modulation and antigen presentation⁶³, our data did not predict nor show synergy of venetoclax with anti-PD-1, which is in line with previous observations⁶⁴. Instead, we suggest that venetoclax might, through optimal, anti-CD40 immune modulation, target the CSC niche. Future research questions include whether there is a direct effect of this targeted therapy on CSCs (Fig. 6a), what is the role of apoptotic priming⁶⁵ and should venetoclax be used as a common combination partner with other drugs to eliminate the resistant CSCs. Panobinostat-associated human data are limited⁶⁶; however, mouse studies using a more specific, class II HDAC inhibitor, in the same MMTV-PyMT model, showed that infiltration of antigen-presenting macrophages is a mechanism of action in anti-PD-1 therapy response⁵². Our data support this observation, as we saw significant infiltration of the same cell type specifically at the panobinostat condition (Fig. 1g). We extend this knowledge and suggest that, in addition to APC infiltration⁵², induction of immunogenic cell death and proficient antigen presentation machinery in general (both tumor-MHC-I⁵¹ and MHC-II on different myeloid cells) might be important attributes of effective induction of anti-tumor immunity in breast cancer, and we suggest that epigenetic modulators in general should be considered for ICB synergy in breast cancer. We also showed that probes ICAM1, calreticulin, PD-L1, neuropilin-1, galectin-3 and MPO were spatially associated with immunogenic cell death and, together with the enriched standard cell types, they might serve as an early predictive biomarker of induced anti-tumor immunity in situ. Although ICB immunotherapies are increasingly well-established for breast cancer^51,52,63,64, anti-CD40 agonists have been evaluated mostly in pancreatic cancer⁶⁷ where their efficacy is party attributed to dense stroma elimination⁶⁸. We observed enrichment of fibroblasts in the CSC niche (Fig. 5e, f). Whether anti-CD40 affects the fibrotic degradation in the niche remains to be determined (Fig. 6a). Nevertheless, considering the strong infiltration of myeloid cells and enrichment of non-immune stroma induced by primary chemotherapies and targeted agents, and the capacity of anti-CD40 to modulate these components to stimulate anti-tumor effects^{42,43,44,68,69,70} (Fig. 6a), perhaps anti-CD40 agonists might be the optimal immunotherapy in breast cancer treatment.