• Breast Cancer
• Tumor microenvironment - TME
• Chemotherapy
• Immunotherapy

Triple-negative breast cancer (TNBC) is known as the most aggressive and refractory breast cancer subtype with limited therapeutic approaches. To date, chemotherapy like paclitaxel (PTX) and nab-paclitaxel (Nab-P) still functions as the bedrock, while immune checkpoint blockade (ICB), antibody-drug conjugates, poly (ADP-ribose) polymerase inhibitors and other promising therapies keep evolving.1 Over the past decade, the combined application of chemotherapy and ICB has reshaped the clinical paradigm of TNBC, but the results turned out to be perplexing: the anti-programmed cell death protein-1 (PD-1) pembrolizumab in combination with chemotherapy has attained official approval in both metastatic and early TNBC based on the KEYNOTE-355 and KEYNOTE-522 trials. As for the anti-programmed cell death-ligand 1 (PD-L1) atezolizumab (ATZ), instead, the IMpassion130 trial acquired positive results and propelled the official approval, but the subsequent IMpassion131 and IMpassion132 trials ended up in failure, leading to withdrawal of the pertinent indication.2 Besides, chemoimmunotherapy only takes effect in a minority of specific TNBC populations. These responsive subgroups are usually regarded as intrinsic “immune-hot”, typically featured by high expression of PD-L1 (only in the metastatic setting) and enrichment of tumor-infiltrating lymphocytes, necessitating further elucidation of the cellular dynamics in the tumor immune microenvironment (TIME) during chemoimmunotherapy, as well as exploration of predictive biomarkers for treatment response.

The emerging single-cell sequencing techniques such as single-cell RNA sequencing, T-cell receptor sequencing and assay for transposase-accessible chromatin sequencing enable comprehensive and detailed delineation of the cellular landscape within the TIME, so that complicated interactions at the single-cell resolution could get incrementally unraveled to interpret the divergent clinical responses to chemoimmunotherapy.3 Back to 2021, Dr Yuanyuan Zhang and colleagues had analyzed immune cell proportions before treatment (termed as predictive index) and post-treatment alterations (termed as therapeutic index) in 22 patients with advanced TNBC treated by PTX with or without ATZ, who found out that the ATZ+PTX combination significantly expanded CXCL13⁺ T cells, conventional type 1 dendritic cells (DCs), lymphoid tissue inducer cells and follicular B (Bfoc) cells in responders, while PTX could reverse the expansion of these immune cells.4 Moving forward from this previous study, the researchers diversified the study cohort, and further deciphered the immune dynamics in TNBC treated by PTX, Nab-P and their respective combination with ATZ. After analyzing 447,494 immune cells from 44 patients and 78 specimens in total, the authors came to the conclusion that in contrast to PTX, Nab-P was capable of recruiting and activating CD8⁺TCF7⁺ stem-like effector memory T cells and CD4⁺ T follicular helper cells, as well as DCs, pro-inflammatory macrophages and various B (including Bfoc, naive B (Bn) and memory B (Bmem)) cells. Such enrichment and enhancement of immune cells was intriguingly attributed to the activation of tumor-infiltrating mast cells (TIMCs), which were found to get bolstered by Nab-P to reprogram myeloid cells like pro-inflammatory macrophages and monocytes through the colony-stimulating factor signaling pathway, and further recruit lymphocytes via the C-X-C motif chemokine ligand 9 (CXCL9)/C-X-C motif chemokine receptor 3 (CXCR3) axis (figure 1).5 Therefore, proper modulation of TIMCs might serve as a promising chemoimmunotherapy sensitization tactic for patients with TNBC, especially for those predicted as non-responders prior to treatment.

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Figure 1

Typical immune dynamics reveal the pro-inflammatory role of TIMCs in patients with TNBC treated by chemoimmunotherapy. The distinct immune alterations in patients with TNBC after treatment of PTX, Nab-P and their combination with ICB were uncovered by single-cell RNA sequencing. It was found that Nab-P-activated TIMCs could promote myeloid cells including pro-inflammatory macrophages and DCs via the CSF pathway, which could further boost T and B lymphocytes to exert their antitumor effects via the CXCL9-CXCR3 axis. The figure was created with figdraw. ATZ, atezolizumab; Bn, naive B cell; cDC, conventional dendritic cell; CSF, colony-stimulating factor; CXCL9, C-X-C motif chemokine ligand 9; CXCR3, C-X-C motif chemokine receptor 3; DC, dendritic cell; ICB, immune checkpoint blockade; mDC, mature or migratory dentritic cell; Nab-P, nab-paclitaxel; PTX, paclitaxel; Tex, exhausted T cell; Tfh, T follicular helper cell; TIMC, tumor-infiltrating mast cell; TNBC, triple-negative breast cancer; Treg, regulatory T cell; Tstr, T cell stress response state.

As a component of the myeloid lineage and innate immune system, the roles of mast cells in allergy and parasite infection have been well understood, but their functions in cancer remain unclear and controversial. Regarded as “few but efficient” in the tumor microenvironment of breast cancer and other solid tumors, TIMCs localize at either marginal or central regions, and exert either protumor or antitumor/inflammatory effects by secreting various cytokines and modulating immune cells depending on the milieu.6 Recent studies are unveiling pivotal mediating effects of TIMCs in tumor proliferation, microenvironment reprogramming and immunotherapy resistance. For instance, Panagi et al indicated that TIMCs could promote the stiffness of the extracellular matrix by proliferating cancer-associated fibroblasts in fibrosarcoma and osteosarcoma. Harnessing the antihistamine ketotifen to suppress mast cell activation could reverse the stiffening process and improve tissue perfusion, hence sensitizing sarcomas to immunotherapy with boosted infiltration and tumor-specific memory of T cells.7 Likewise, CXCR2⁺ TIMCs with inhibited ferroptosis and enhanced proliferation could get recruited by pancreatic ductal adenocarcinoma (PDAC) cells. These tumor-associated mast cells could produce CXCL10 to promote epithelial-mesenchymal transition (EMT) and create suppressive TIME by enriching CXCR3⁺ regulatory T (Treg) cells. Consistently, the mast cell membrane stabilizer sodium cromoglycate was proven to compromise CXCL10 and further augment the efficacy of anti-PD-L1 plus gemcitabine in an orthotopic murine model of PDAC.8

Zooming into breast cancer, depending on the expression of hormone receptors and human epidermal growth factor receptor 2 (HER2), it is widely acknowledged that breast cancer is stratified as several molecular subtypes with distinct TIME contexts and clinical implications.9 Of note, the density of TIMCs and their correlation with clinical prognosis even varies between different breast cancer subtypes: TIMC infiltration is more prevalent in the luminal subtype as compared with HER2-positive and TNBC, and usually indicates worse response in HER2-positive breast cancer but better in the case of TNBC,9 the latter of which is in line with the latest discovery.5 However, some other studies arrived at opposite conclusions that TIMCs were linked to the inverse survival predictor Annexin A1, as well as several protumor phenotypes like angiogenesis and EMT in basal-like or TNBC,6 suggesting the heterogeneity and intricacy of this specific cell population.

Grounded on the obtained knowledge, targeting TIMCs represents a thriving therapeutic direction in cancer, which has been examined in numerous preclinical tumor models.6 Most of the current TIMC-targeting strategies are designed to refrain the activity of mast cells, which can be at least classified as (1) mast cell degranulation stabilization by cromolyn, nedocromil or lodoxamide; (2) modulation of mast cell membrane receptors (eg, PD-L1, toll-like receptor and sialic acid-binding Ig-like lectin 8) and/or secreted mediators (eg, histamine, tryptase and tumor necrosis factor α) by small-molecular inhibitor or monoclonal antibody (mAb); (3) mast cells reduction by tyrosine kinase inhibitors like imatinib, dasatinib or sunitinib to refrain c-Kit activity, since mast cells can be stimulated by stem cell factor via c-Kit.10 In contrast, pharmacological activation of mast cells in specific tumor types like TNBC, where TIMCs might exert pro-inflammatory and antitumor effects, was scarcely focused on before.

Considering the discerned favorable role of TIMCs in the context of TNBC, the researchers compared the results between bidirectional modulations of TIMCs.5 Initially, they administered the mast cell degranulation promoter compound-48/80 trihydrochloride (C48/80), aiming to explore the outcome of TIMC activation in the TIME of TNBC. Despite limited impact on tumor growth, the enrollment of C48/80 did give rise to increased mast cells as well as T-cell populations, including naive, helper (CD4⁺CXCR5⁺), effector and exhausted (CD8⁺GZMB⁺) T cells, the latter of which may account for the disfavored antitumor effect. In terms of B-cell subsets, C48/80 could ignite CD19⁺CD27⁺ Bn and Bmem cells, while Bfoc cells were expanded in ATZ+Nab-P responders. On the contrary, Nab-P and anti-PD-L1 mAb were also applied with or without disodium cromoglycate (DSCG, a mast cell degranulation stabilizer) in the mouse model. It was found that the addition of DSCG significantly weakened tumor shrinkage and upregulated suppressive DCs, cementing again the pivotal tuning roles of TIMCs underlying chemoimmunotherapy.

Collectively, Zhang et al systematically analyzed single-cell alterations in the TIME of TNBC, and pointed out that TIMC activation might hold promise for efficacy enhancement in synergy with chemoimmunotherapy. Despite these inspiring and enlightening findings, a few limitations and unanswered questions remain to be noted and resolved. As was mentioned in the discussion section,5 the total sample size is expected to be further enlarged in the pursuit of enhanced comparability and controlled variability, so are paired samples between primary and metastatic lesions, as well as standardized evaluation of treatment responses. In addition, how to design a specific, effective and safe TIMC agonist for patients with TNBC? Would pharmacological activation of TIMCs increase the risk of allergic reactions and other potential adverse events in the clinical setting? How to employ tailored TIMC-targeting therapy and identify reliable biomarkers in other tumor types beyond TNBC, where TIMCs might exhibit divergent TIME-orchestrating characteristics? To our delight, a phase II clinical trial (NCT05076682) has been initiated in Fudan University Shanghai Cancer Center, China. This ongoing trial aimed to reverse ICB resistance in metastatic TNBC by exploring the efficacy and safety of intranasal sodium cromoglicate, choline or efavirenz in combination with chemoimmunotherapy. However, given the highly heterogeneous, TIME-dependent phenotype plasticity of TIMCs, as well as the absence of clinical evidence by now, more works are warranted before this burgeoning conception comes true.