What if a chemotherapy drug does more than shrink a tumor? In this melanoma study, doxorubicin also helped organize immune cells into tertiary lymphoid structures—while increasing a checkpoint signal that could shape combination therapy. The tumor immune microenvironment, or TIME, comprises abundant immune cells and plays an important role in the antitumor response.

Tertiary lymphoid structures are organized aggregates of immune cells that form postnatally in non-lymphoid tissues of the TIME. These structures are composed of high endothelial venules and a variety of immune cells, and are considered the local site where antigen-specific CD8+ T cells are generated in the tumor microenvironment.

Their presence is associated with better prognosis and clinical outcomes, including in melanoma, and can serve as a predictive indicator of clinical efficacy in immune checkpoint inhibitor immunotherapy. Yet no studies had explored how different chemotherapeutic drugs affect TLS in the tumor microenvironment.

Chemotherapeutic drugs can be divided into immunogenic cell death, or ICD, and non-ICD drugs based on whether they trigger an immune response. When ICD occurs, dying cells produce new antigenic epitopes and release damage-associated molecular patterns, such as calreticulin, HMGB1, and ATP.

Those signals recruit antigen-presenting cells to recognize and present antigens on dying cells to T cells, establishing a persistent antitumor immune effect by activating the immune response system to eliminate tumor antigens. But whether ICDs induce tumor-localized high endothelial venules and tertiary lymphoid structures remains unclear.

Doxorubicin is a representative anthracycline drug that mainly acts on DNA to exert cytotoxicity and serves as a representative chemotherapeutic drug for ICD. Gemcitabine is a cytidine analog that inhibits DNA synthesis. Although gemcitabine can increase calreticulin exposure and HMGB1 release in vitro, it inhibits damage-associated molecular patterns by triggering prostaglandin E2 in vivo.

Female C57BL/6 mice aged between six and eight weeks were used, and B16F1 cells were inoculated subcutaneously into the mice at a density of five times ten to the fifth. Nine days after inoculation, the mice were divided into four groups: PBS, doxorubicin, gemcitabine, and combined treatment.

The corresponding treatments were PBS at one hundred microliters per mouse, doxorubicin at five milligrams per kilogram, gemcitabine at twenty-five milligrams per kilogram, and a combination of doxorubicin and gemcitabine for six days. After treatment began, tumor length and width were measured daily, and tumor volumes were quantified as length times width times height divided by two.

Figure one outlines the melanoma experiment: B16F1 cells were implanted in mice, treatments began on day nine, and tumors were collected on day seventeen. The tumor photographs and volume curves show the outcomes for PBS, doxorubicin, gemcitabine, and combined treatment, while panels D and E assess HMGB1 staining using representative immunohistochemistry images and a combined percentage-and-intensity score.

Together, the figure links chemotherapy with tumor-growth measurements and altered HMGB1 expression, motivating the immune analyses that follow. The melanoma mouse model was established by subcutaneously inoculating B16F1 tumor cells into the groin region of C57BL/6 mice.

The detailed administration schedule and tumor harvest intervals are shown in Figure 1A. Tumor volumes were monitored daily, the data were plotted, and harvested tumors were photographed. To study changes in the immune microenvironment after doxorubicin treatment, single-cell suspensions were prepared from tumor tissues for flow cytometry analysis.

The proportion of CD3+ T cells in the doxorubicin group and combination-treatment group was significantly higher than in the gemcitabine group. Doxorubicin alone significantly promoted infiltration of CD8+ T cells compared with control and gemcitabine treatments, but it did not have a significant effect on CD4+ T cells.

The study next analyzed the levels of cytotoxic cytokines secreted by CD4+ T cells and CD8+ T cells. Figure two uses flow cytometry on melanoma tumors harvested at day seventeen to profile T-cell populations after PBS, doxorubicin, gemcitabine, or combination treatment.

The plots and summary charts measure total CD3-positive cells, CD4 and CD8 subsets, functional markers including interferon gamma, granzyme B, and perforin, plus Foxp3-positive regulatory T cells and CD103-positive cells. Notably, the authors report increased T-cell infiltration with doxorubicin-containing treatment and reduced Foxp3-positive fractions across all treatment groups, linking doxorubicin to changes in the tumor immune environment.

Figure three uses flow cytometry to track antigen-presenting cells across PBS, doxorubicin, gemcitabine, and combination treatment groups, with five samples per group. Doxorubicin increased the gated B-cell and dendritic-cell populations to thirty-six point three percent and thirty-six point nine percent, respectively, compared with seventeen point eight percent and fifteen point seven percent in the control group.

The CD86 panels also assess activation, showing increased CD86-positive B cells and dendritic cells after doxorubicin, supporting enhanced immune-cell recruitment and activation in the tumor tissue. High endothelial venules are the main portal for lymphocytes entering tumor tissues and the most important component of tertiary lymphoid structures.

The proportion of PNAd+ high endothelial venules in CD45- cells was measured by flow cytometry. That proportion was significantly higher in the doxorubicin group than in the control, gemcitabine, and combination-treatment groups. The density of PNAd+ high endothelial venules was measured in the samples to quantify tertiary lymphoid structure formation.

Doxorubicin treatment significantly promoted tertiary lymphoid structure formation compared with control, gemcitabine, and combination treatment. These data were consistent with the flow cytometry results. Figure four links doxorubicin treatment with HEV and TLS-associated immune organization.

In panel A, PNAd-positive cells comprise seven point two three percent with PBS, nineteen point six percent with doxorubicin, eight point seven one percent with gemcitabine, and ten point five percent with the combination. Immunofluorescence in panel B marks T cells, B cells, PNAd, and nuclei, while panels C through E quantify T-cell and B-cell occupancy and PNAd-positive HEV density across five fields per sample.

Doxorubicin significantly increased PD-1 expression on CD8+ T cells compared with the control and combination treatments. No statistically significant differences were found in PD-1 expression on CD4+ T cells among the four groups. Figure five measures checkpoint expression by flow cytometry across PBS, doxorubicin, gemcitabine, and combination groups.

In panel A, doxorubicin is associated with a larger PD-one-positive fraction among CD-eight T cells, while the figure reports no significant difference for CD-four T cells. Panel B shows PD-L-one staining in CD-forty-five-negative tumor cells, with values of six point three seven percent for PBS, twenty-two point six percent for doxorubicin, nine point seven eight percent for gemcitabine, and fourteen percent for the combination.

Immunogenic cell death induction in tumor cells is described as a promising approach for activating antitumor immune responses. Chemotherapeutics that promote ICD can recruit and promote dendritic-cell maturation and cross-initiation of tumor-specific CD8+ T cells through damage-associated molecular pattern release.

HMGB1 plays a crucial role in this process, and by binding to toll-like receptor four on dendritic cells, HMGB1 promotes dendritic-cell maturation and releases pro-inflammatory cytokines to trigger an effective immune response. In this study, doxorubicin treatment promoted infiltration and activation of dendritic cells and enhanced the function of infiltrated CD8+ T cells, including expression of interferon-gamma, granzyme B, and perforin.

Although doxorubicin promoted immune-cell infiltration and tertiary lymphoid structure formation simultaneously, the study still has limitations. Only one ICD inducer was used, and the results were examined only in a mouse model of melanoma.

Additional chemotherapeutics with ICD reactions and additional experimental models should be used for further confirmation. The conclusion is that doxorubicin, with an ICD reaction, promoted tertiary lymphoid structure formation and increased PD-1 and PD-L1 expression in tumor tissues, which may be advantageous for combined immune checkpoint therapy.

In this mouse melanoma model, doxorubicin increased immune-cell infiltration, promoted HEV and TLS formation, and increased PD-1 and PD-L1 expression. The result supports testing ICD-inducing chemotherapy with checkpoint therapy, but only one drug and one model were studied.