3.1 Screening the possible immunity functions related anti-cancer treatment.
Nearly all implicated immune checkpoints were browsed and screened for possible immunotherapies values, and the representative immunity therapeutics of ADORA2A, BTLA, Nectin-2 (CD112), CD160, CD244, PD-L1 (CD274), CD96, CSF1R, CTLA4, HAVCR2, IDO1, IL10, IL10RB, KDR, KIR2DL1, KIR2DL3, LAG3, LGALS9, PDCD1, PDCD1LG2, PVRL2, TGFB1, TGFBR1, TIGIT, VTCN1, TNF Receptor Superfamily Member 14 (TNFRSF14), TNF superfamily member 4 (TNFSF4), TNF superfamily member 18 (TNFSF18), were all input for possible effects predication in the integrated repository portal for tumor-immune system interactions systems at TISIDB [15]. Primarily, the PD-L1, CD112, TNFRSF14, TNFSF4, TNFSF18, CD48, and LGALS9 were selected for their significantly and differently expressed patterns, and the illustrating body-map was shown in Fig. S1, the paired red and green color indicated the expression patterns in multiple organs and systems.
Further, the PD-L1 (Fig. 1A), CD112 (Fig. 1B), TNFRSF14 (Fig. 1C), TNFSF4 (Fig. 1D), TNFSF18 (Fig. 1E), CD48 (Fig. 1F), and LGALS9 (Fig. 1G) were analyzed for their abnormally expressed styles in breast carcinomas. Specifically, CD112, TNFSF4, TNFSF18, and LGALS9 were significantly overexpressed in breast carcinomas, strongly suggested the potent and valuable effects.
3.2 Immunotherapeutic targets participated in multiple breast carcinogenesis through interacting with the key cancer stimulating factors
The heterogenetic features of breast cancer determined that breast carcinomas of different hormone receptors status may benefit from different and specific treating strategies. As a double-edged sword, one specific agent will not function in another kind of breast carcinoma, and to dig out the potentials of immune blockades therapies, the native expression signatures and correlations were studied through using the CBIOPORTAL and the GEPIA2 (GENE EXPRESSION PROFILING INTERACTIVE ANALYSIS). The expression heatmap indicated the expression patterns of the candidates in red bar, and the selected representative genes described in section 3.1 were labeled with RED STAR (Fig. 2A), and the analyzing chart flow was shown in Fig. 2B. All the enrolled factors were input and studied for potential functional correlations (Fig. S2). Specifically, the main carcinogens of ERBB2, KRAS, TP53 were analyze for their intrinsic connection in breast carcinomas, and the promising correlations between TNFSF4 and the ERBB2, KRAS, TP53 were all confirmed (Fig. S3, Table 1).
In general, nearly all the immunotherapeutic targets showed expanded expression intervals referring to ESR1 and PGR, two of which tended to react well to formal and anti-hormone treatments, indicating that immunotherapy may compensate the current deficiencies. More importantly and interestingly, TNFSF4, TNFSF18, CD48, all showed aggregated expressions in breast carcinomas bearing no ERBB2, ESR1, PGR expression, strongly suggested that the immune therapy may cover all types of breast cancer, and the partial enlarged drawing was shown in Fig. 2C. In detail, TNFSF4, TNFSF18 and CD48 showed exotic expression to all kinds of breast cancer carcinogens, including those without KRAS (Fig. 2D), TP53 (Fig. 2E), ERBB2 (Fig. 2F) expressing and activating, the three of which dominated the survival expectance and drawn the dark survival expectance.
3.3 Evaluation of the cluster of TNFSF4, TNFSF18, CD48 and LGALS9 as the immune therapeutics targets.
The clinical significance of the cluster of TNFSF18 (Fig. 3A), LGALS9 (Fig. 3B), TNFSF4 (Fig. 3C) and CD48 (Fig. 3D) were also analyzed for their values referring to immune therapeutics targets, and the TNFSF4, TNFSF18 showed greater significances in predicating the disease-specific survival and the overall survival. KRAS stimulated the carcinogenesis greatly and determined the anti-cancer treatment response, we further explored its correlation with TNFSF18 (Fig. 3E), LGALS9 (Fig. 3F), TNFSF4 (Fig. 3G) and CD48 (Fig. 3H), and the positive correlation between TNFSF4 and KRAS strongly suggested the prospect therapy effects of using TNFSF4 as the manipulating or the blocking targets.
Stem cells groups were considered as the root for cancer recurrence for their steady status and super renewal ability. We previously identified the stem-like ALDH1A1+ cells in breast cancer groups [16-18], and in this study, we noticed the close correlation between TNFSF4 and ALDH1A1 expressions (Fig. 3I), the latter of which indicated the poorer survival length (Fig. 3J) and progression free length (Fig. 3K). These results strongly proved that the oncogenic TNFSF4 may be one effective immunotherapeutic target, and the proposed survival benefits may also function through repressing the stem cells expansion, fully assure the anti-TNFSF4 treatments’ results. The other candidates of CD112, TNFRSF14, PD-L1 failed to be involved in further analysis, as their negative survival indicating roles (Fig. S4).
3.4 The mechanistic procedure that TNFSF4 was assumed to function through
The immune system was locked when carcinoma is getting aggressive, and when the immune-function inhibitors were unlocked (immune blockades), the active immune cells begin to infiltrate and execute the cellular order (Fig. 4A). The whole cancer groups, including the heterogenetic subtype cells of cancer stem cells, will be perished under the immune system attack. To undermine the crucial role of the selective immune target, the lymphocytes infiltrating functions and the connective functional factors were analyzed respectively for underlining the mechanisms. Both the ALDH1A1 (Fig. 4B) and TNFSF4 (Fig. 4C) overexpression correlated with more active lymphocytes. However, the guardian soldiers were disabled with the highly expressed immune-inhibitors in cells with increased ALDH1A1 (Fig. 4D) and TNFSF4 (Fig. 4E). These results indicated that TNFSF4 will potentially reactive the immune response, and will partially function through precisely demilitarize the stem cells.
3.6 Promising immune therapies response are the stem cells signatures associated predicating mode
The immune therapies response and effects were believed to be correlated with lymphocytes activation and infiltration, and we first assessed the immune therapies effects in multiple systems, treatments targeted at TNFSF4 tended to implicit the better outcomes (Fig. 5A). Also, the TNFSF4 associated TP53 (Fig. 5B), KRAS (Fig. 5C), and ERBB2 (Fig. 5D) all indicated better immune therapy response, which further consolidated the crucial position of TNFSF4.
TNFSF4 and ALDH1A1 were confirmed for their participants in immune activation therapies, and TNFSF4 treatment predicated the prospect therapy response. Immune therapy consistently repressed the tumor growth once the immune system was activated, and in above results we further found that TNFSF4 associated therapy may also influence the stem cells expansion. Increased ALDH1A1 expression indicated shorter disease specific survival (Fig. 5E) and overall survival (Fig. 5F), and in clinical assessing, and the ALDH1A1 surprisingly correlated with higher therapy responds ratios (Fig. 5G), which has not ever been reported and analyzed.
Associations between ALDH1A1 expression and immune subtypes across human cancers were all included for analysis, and ALDH1A1 expression dominated in all the subtypes (Fig. 5H), participating in multiple immune reaction process (Fig. 5I). Associations between ALDH1A1 expression and molecular subtypes across human cancers were also identified (Fig. 5J), and the signature of increasing ALDH1A1 tended to express in all kinds of breast carcinomas (Fig. 5K), indicating the universal therapeutic responsive role of ALDH1A1 referring to better outcomes.