*3.5. Combination of TLM\_CFM-F\_OSM Induces Anti-Cancer Effect via the LAMIN B2/STAT3/NF-κB Signaling Pathways in Lung Cancer*

Western blot analysis was performed to evaluate the protein expression of Lamin B2, SOD, STAT3, and NFKB. TLM\_CFM-F\_OSM treatment group significantly reduced the expression of Lamin B2 when compared to the control (*p* < 0.0001). TLM\_CFM-F\_OSM significantly downregulated the expression of Lamin B2 as compared to TLM\_CFM-S\_OSM (*p* < 0.01) and single treatment groups OSM (*p* < 0.05), CFM-F (*p* < 0.01). Further, the TLM\_CFM-F\_OSM combination significantly decreased the expression of SOD2 (*p* < 0.01), NFκB (*p* < 0.0001), STAT3 (*p* < 0.0001) protein as compared to control and single treatments (CFM-F). Collectively TLM\_CFM-F\_OSM demonstrated a superior anti-cancer effect, thereby decreasing the protein expression of lamin B2, SOD, STAT3, and NFKB (Figure 4). *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 12 of 21

**Figure 4.** Western blot data analysis for the effect of Telmisartan, CFM-F, and Osimertinib and their combinations against H1975 lung cancer. Western blots and densitometric analysis of various proteins in the H1975 xenograft model of lung cancer. Data are representative of three different experiments and presented as mean, and error bars refer to SEM. ns *p* > 0.05 \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 was considered significant when compared to the control. **Figure 4.** Western blot data analysis for the effect of Telmisartan, CFM-F, and Osimertinib and their combinations against H1975 lung cancer. Western blots and densitometric analysis of various proteins in the H1975 xenograft model of lung cancer. Data are representative of three different experiments and presented as mean, and error bars refer to SEM. ns *p* > 0.05 \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 was considered significant when compared to the control.

#### *3.6. Combination of TLM\_CFM-F\_OSM Reduced the Protein Expression of Lung Cancer Stem Cells, Fibrosis, and Migration 3.6. Combination of TLM\_CFM-F\_OSM Reduced the Protein Expression of Lung Cancer Stem Cells, Fibrosis, and Migration*

We examined the effects of CFM-F, TLM, and OSM on lung cancer stem cell (SOX2), migration (MMP1), and fibrosis (TGF-β) markers, since cancer stem cells, fibrosis and migration play a certain role in drug resistance development and cancer cell proliferation. It was observed that CFM-F, OSM, and TLM alone groups did not reduce the expression of TGF-β, MMP1, Oct 4, and Sox2 in H1975 lung tumors. The combination of TLM\_CFM-F\_OSM significantly reduced the protein expression of TGF-β (*p* < 0.0001), Sox2 (*p* < 0.0001), and MMP1 (*p* < 0.01) levels in H1975 xenografts as compared to control and TLM\_CFM-S\_OSM. Pre-treatment with CFM-F, followed by therapy with OSM, resulted in a significant reduction in the expression of lung cancer stem cell markers, migration, and fibrosis. Pre-treatment with TLM and CFM-F enhanced OSM sensitivity (Figure 4). We examined the effects of CFM-F, TLM, and OSM on lung cancer stem cell (SOX2), migration (MMP1), and fibrosis (TGF-β) markers, since cancer stem cells, fibrosis and migration play a certain role in drug resistance development and cancer cell proliferation. It was observed that CFM-F, OSM, and TLM alone groups did not reduce the expression of TGF-β, MMP1, Oct 4, and Sox2 in H1975 lung tumors. The combination of TLM\_CFM-F\_OSM significantly reduced the protein expression of TGF-β (*p* < 0.0001), Sox2 (*p* < 0.0001), and MMP1 (*p* < 0.01) levels in H1975 xenografts as compared to control and TLM\_CFM-S\_OSM. Pre-treatment with CFM-F, followed by therapy with OSM, resulted in a significant reduction in the expression of lung cancer stem cell markers, migration, and fibrosis. Pre-treatment with TLM and CFM-F enhanced OSM sensitivity (Figure 4).

*3.7. Combination of TLM\_CFM-F\_OSM Effects on the Protein Expression of Tumor Suppressor* 

Bax (*p* < 0.0001) in the H1975 tumors as compared to control. Here, the combination of TLM\_CFM-F\_OSM leads to the induction of apoptosis by increasing the protein expres-

*3.8. The Effects of TLM\_CFM-F\_OSM Combination on Exosomal Markers Expression in H1975* 

The tumor suppressor proteins (p38 and p53), AMPK, and the Bax protein all appear

sion of p38, p53, AMPK, and Bax in H1975 lung tumors (Figure 4).

*Proteins and Apoptotic Proteins* 

*Lung Cancer* 

#### *3.7. Combination of TLM\_CFM-F\_OSM Effects on the Protein Expression of Tumor Suppressor Proteins and Apoptotic Proteins*

The tumor suppressor proteins (p38 and p53), AMPK, and the Bax protein all appear to affect cell apoptosis susceptibility. The combination of TLM CFM-F OSM significantly enhanced protein expression of p38 (*p* < 0.0001), p53 (*p* < 0.0001), AMPK (*p* < 0.0001) and Bax (*p* < 0.0001) in the H1975 tumors as compared to control. Here, the combination of TLM\_CFM-F\_OSM leads to the induction of apoptosis by increasing the protein expression of p38, p53, AMPK, and Bax in H1975 lung tumors (Figure 4). *Pharmaceutics* **2022**, *14*, x FOR PEER REVIEW 13 of 21

## *3.8. The Effects of TLM\_CFM-F\_OSM Combination on Exosomal Markers Expression in H1975 Lung Cancer*

Exosomes (EVs) are intercellular messengers that play a key role in cancer formation. CD63, TSG 101, Flotilin-2, calnexin, Syntenin-1, and Caveolin-1 are commonly used exosomal markers. TotalSeq assay from mouse serum EVs revealed that TLM\_CFM-F\_OSM induced the downregulation of exosomal marker CD63, CD81, and oncogenic proteins (EGFR and PD-L1) in lung cancer, and we further validated these results by checking the protein expression through Western blotting in lung cancer tissues after their respective treatments. Exosomal markers CD63 (*p* < 0.0001), TSG 101 (*p* < 0.001), Flotillin-2 (*p* < 0.0001), Calnexin (*p* < 0.0001), Syntenin-1 (*p* < 0.0001) were significantly downregulated and Caveolin-1 (*p* < 0.0001), HSC 70 (*p* < 0.001) protein expression were significantly upregulated as compared to control group as shown in Figure 5. Exosomes (EVs) are intercellular messengers that play a key role in cancer formation. CD63, TSG 101, Flotilin-2, calnexin, Syntenin-1, and Caveolin-1 are commonly used exosomal markers. TotalSeq assay from mouse serum EVs revealed that TLM\_CFM-F\_OSM induced the downregulation of exosomal marker CD63, CD81, and oncogenic proteins (EGFR and PD-L1) in lung cancer, and we further validated these results by checking the protein expression through Western blotting in lung cancer tissues after their respective treatments. Exosomal markers CD63 (*p* < 0.0001), TSG 101 (*p* < 0.001), Flotillin-2 (*p* < 0.0001), Calnexin (*p* < 0.0001), Syntenin-1 (*p* < 0.0001) were significantly downregulated and Caveolin-1 (*p* < 0.0001), HSC 70 (*p* < 0.001) protein expression were significantly upregulated as compared to control group as shown in Figure 5.

**Figure 5.** Analysis of exosomal markers in H1975 xenografts and serum samples: (**A**) Bar graph represents the TotalSeq assay in serum samples of H1975 tumor bearing athymic nude mice after treatment with different drugs and their combinations; (**B**) Representative Western blots showing expressions of exosomal protein markers after treatment in H1975 tumor tissue homogenates; (**C**) bar graphs represent the densitometric analysis of respective Western blots. Data represented as three different experiments and presented as mean ± SEM. ns *p* > 0.05 \*\* *p* < 0.01, and \*\*\* *p* < 0.001 was considered significant when compared to control. **Figure 5.** Analysis of exosomal markers in H1975 xenografts and serum samples: (**A**) Bar graph represents the TotalSeq assay in serum samples of H1975 tumor bearing athymic nude mice after treatment with different drugs and their combinations; (**B**) Representative Western blots showing expressions of exosomal protein markers after treatment in H1975 tumor tissue homogenates; (**C**) bar graphs represent the densitometric analysis of respective Western blots. Data represented as three different experiments and presented as mean ± SEM. ns *p* > 0.05 \*\* *p* < 0.01, and \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001 was considered significant when compared to control.

Lung cancer with EGFR gene mutations has been observed in over 15% of NSCLC

activation or mutations in a variety of oncogenic proteins (in various cancers), including EGFR, fibroblast growth factor receptor, BRAF, MET, Anaplastic lymphoma kinase, vascular endothelial growth factor receptor (VEGFR), and tyrosine-protein kinase Src [54– 56]. Different EGFR-dependent and EGFR-independent mechanisms are responsible for developing the third-generation OSM (TKI) resistance in EGFR mutant NSCLC, and the molecular mechanisms underlying TKI resistance are still being investigated [57–59]. There is a need to combat OSM resistance in NSCLC, and presently, there are very limited

**4. Discussion** 

#### **4. Discussion**

Lung cancer with EGFR gene mutations has been observed in over 15% of NSCLC adenocarcinomas, with a frequency of around 62 percent in Asian populations [4,5,53]. TKIs are the first-line treatment for NSCLC, though resistance can develop due to hyperactivation or mutations in a variety of oncogenic proteins (in various cancers), including EGFR, fibroblast growth factor receptor, BRAF, MET, Anaplastic lymphoma kinase, vascular endothelial growth factor receptor (VEGFR), and tyrosine-protein kinase Src [54–56]. Different EGFR-dependent and EGFR-independent mechanisms are responsible for developing the third-generation OSM (TKI) resistance in EGFR mutant NSCLC, and the molecular mechanisms underlying TKI resistance are still being investigated [57–59]. There is a need to combat OSM resistance in NSCLC, and presently, there are very limited options. Hence, finding new therapeutic targets and treatment options that enhance TKI anti-cancer effects while also overcoming TKI resistance is a critical clinical need for NSCLC. To our knowledge, this is the first study to demonstrate that a combination of TLM, CFM 4.17F, and OSM has anti-cancer potential in H1975 xenografts of athymic nude mice and to understand their mechanisms using proteomics and RNA-seq studies.

The formulation of CFM 4.17 in a lipid formulation has already been established in our laboratory, and we have already demonstrated that a combination of OSM and CFM4.17 formulation inhibited the growth of H1975 cells in vitro more effectively than OSM alone, with an IC<sup>50</sup> value that was 2-fold lower [6]. We did not conduct any in vitro studies for these studies since we have already published extensively with them in an earlier communication [6].

Our in vivo studies revealed that the TLM\_CFM-F\_OSM formulation outperformed CFM-F and CFM-S, as well as their combinations with TLM, in terms of reducing tumor burden. Based on our earlier reports, these observations are not surprising but were expected [6]. For example, in our laboratory, we demonstrated that TLM, when used in combination with CFM 4.16 and Sorafenib, significantly enhanced the anti-cancer effects of sorafenib in rociletinib resistant NSCLC xenografts (*p* < 0.01), which was attributed to disrupting tumor-stromal barriers, allowing sorafenib to penetrate deeper into tumors when administered in vivo [35]. However, in the present study, we used RNA-seq and proteomic analysis to uncover molecular changes in genes and proteins in H1975 cells to investigate the possible anti-cancer mode of action of the triple combination (TLM\_CFM-F\_OSM). RNAseq (KEGG Pathway) revealed differential regulation of several genes, including metabolic (AMPK), immunological (PD-L1), mitochondrial function (SOD), inflammatory pathways (NFKB, STAT3, TGF beta), and apoptotic pathways (Lamin-B2, Macrophage mannose receptor 1). In the treatment of lung cancers, these pathways are linked to adhesion, invasion, evasion, proliferation, migration, differentiation, angiogenesis, apoptosis, and resistance to growth suppressors. In addition, we performed proteomics in the same samples to confirm the RNA-seq results and identified the upregulated (AMPK, P53, P38 and BAX) and downregulated (Lamin B2, STAT3, BCL2, SOX2, MMP-1, PDL-1, SOD, NFKB, TGF beta, and C-Myc) proteins in a sequence that matched the gene expression pattern shown in RNA sequencing data.

Adenosine monophosphate kinase (AMPK) is a bioenergetic sensor that activates in response to an elevation in the AMP/ATP ratio and is phosphorylated at Thr172 of the catalytic subunit by upstream kinases, such as liver kinase B1 (LKB1) or calmodulin kinase 1 (CAMK1), regulating metabolic homeostasis in the cell [60–63]. AMPK activation has been shown to induce tumor regression in neuroblastoma, B-cell chronic lymphocytic leukemia and breast and prostate cancer cells. This is attributed to the multiple signaling pathways, including mTOR inhibition to block protein synthesis, stabilizing p53, and cyclin-dependent kinase inhibitors to induce cell cycle arrest [64]. Similarly, we observed increased AMPK gene and protein expression levels in H1975 lung cancers in vivo after TLM, OSM, and CFM-F, and their combination showed the most superior efficacy in upregulating AMPK expression. OSM stimulates apoptosis, activating the AMPK pathway in colorectal cancer cells [65]. TLM has been demonstrated to inhibit cell growth by inducing apoptosis in

a variety of cancer cell lines, including hepatocellular carcinoma (HLF cells) and gastric cancer (MKN45 cells) [66,67]. Our earlier studies have demonstrated that CFM-1, -4, and -5, CARP-1 functional mimetics, suppressed malignant pleural mesothelioma cell growth by triggering apoptosis in vitro. Further, CFM4.16 has been demonstrated to cause apoptosis via activating pro-apoptotic stress-activated protein kinases (SAPKs) such as p38 and JNK and enhanced CARP-1 production loss of the oncogene c-myc, PARP1 cleavage, and mitotic cyclin B1 [68,69]. However, while CFM 4.17-F treatment increased AMPK expression, the specific mechanism behind its activation of AMPK has to be further investigated through knockdown or knock-in studies. Based on these findings, we hypothesize that TLM\_CFM-F\_OSM induces apoptosis in H1975 tumors by activating AMPK.

MAPK pathways have been shown to regulate cancer growth and progression by modulating gene expression, mitosis, proliferation, metabolism, and apoptosis [70]. AMPK activity has recently been linked to p38 MAPK in several studies. AICAR, an AMPK activator that activates the p38 MAPK pathway, increases glucose uptake in skeletal muscle, but the p38 MAPK inhibitor did not affect AICAR activation [71]. Furthermore, AMPK phosphorylation of P38 MAPK induces P53 protein in various cancer cells [72]. Many cancer studies have shown that the AMPK/P38/P53 pathway increases apoptosis by regulating the expressions of BAX and caspases [73,74]. In the current study, H1975 xenografts treated with the TLM\_CFM-F\_OSM combination had higher levels of AMPK, p38, p53, and Bax proteins (Figure 4). Further, TLM\_CFM-F\_OSM significantly reduced cell proliferation and induced apoptotic cell death in H1975 lung tumors via the AMPK/p38 pathway in a p53-dependent manner compared to other treatments.

The intermediate filaments, known as Lamins, line the inner nuclear membrane, provide structural support for the nucleus and regulate gene expression [75]. Lamin B2 promotes the malignant phenotype of non-small cell lung cancer cells by interacting with micro chromosome maintenance protein 7 and Cyclin D1, both of which increase tumor motility and tumor cell epithelial-mesenchymal transition [76]. In a Drosophila laminopathy model, Chandran et al. demonstrated activating AMPK suppresses Lamin mutations and thus regulates laminopathies [77]. However, the precise relationship found between Lamin B2 and AMPK is unknown in cancer studies, and only very few studies have been conducted to investigate the role of Lamin B2 in cancer progression. The most interesting finding in our study is that Lamin B2 is the most differentially expressed protein and is highly under-expressed in the TLM\_CFM-F\_OSM combination treatment. Based on these findings, we hypothesize that AMPK activation by TLM\_CFM-F\_OSM combination controls Lamin B2 expression and thus cell proliferation, migration, and invasion in H1975 tumors.

The pleiotropic transcription factor nuclear factor-κB (NF-κB) influences lung carcinogenesis, thereby upregulating genes involved in cell proliferation, metastasis, cell migration, invasion, and apoptosis suppression [78]. Although tissue heterogeneity exists in lung cancers, the samples collected from the patients always showed an increased level of NF-κB in NSCLC [79]. In line with these findings, we discovered increased NF-κB gene and protein expression levels in H1975 tumors in the current study. Interestingly, the combination of TLM, CFM 4.17, and OSM outperformed their individual treatments in decreasing NF-κB expression in H1975 tumors. In a recent study, Jiang et al. revealed that NCI-H1975/OSM-resistant cells were highly dependent on the NF-κB pathway for survival; the treatment with the NF-κB pathway inhibitor BAY 11-7082 or genetic silencing of p65 resulted in a significantly greater number of cell deaths when compared to parental NCI-H1975/OSM resistant cells [80]. The same study demonstrated that OSM resistance was achieved through TGFβ2-mediated epithelial-mesenchymal transition and NF-κB pathway activation. In our laboratory, we have already demonstrated that CFM-4.16 formulation in combination with sorafenib inhibited the growth of tumor xenografts formed from rociletinib-resistant H1975 NSCLC cells by inhibiting the NF-κB pathway [35] and TLM treatment significantly reduced the inflammatory and hyperproliferative changes in lung tissue after ovalbumin challenge in rats [81]. Based on these findings, we hypothesize that

TLM and the CFM 4.17F increase OSM antitumor effects in H1975 cancers by decreasing NF-κB activity.

TGF-β regulates the proliferation, differentiation, apoptosis, migration, adhesion, immune surveillance, and survival of many cancer cells. According to Mingze ma et al., TGF-β promotes epithelial-mesenchymal transition in A549 human lung cancer cells via the NF-κB/nox4/ROS signaling pathway [82]. In line with these findings, the current study demonstrated considerable TGF-β expression in H1975 tumors and treatment with the TLM\_CFM-F\_OSM combination significantly reduced its expression in H1975 tumors, as evidenced by Western blotting, RNA sequencing, and proteomics analysis. TLM reduced TGF-β levels in NSCLC lung tumors, which was linked to PPAR-γ activation, VEGF, and MMP-9 inhibition, resulting in more nanoparticle penetration into the tumor [28,31,32]. As a result, TLM, in combination with any other anti-cancer drug, would be more effective in treating metastatic lung cancers. In addition, MMP-9 is involved in lung cancer invasion, metastasis, angiogenesis, and progression [83]. It negatively affects cancer immune modulation via TGF-β activation and intercellular adhesion molecule-1 shedding (ICAM-1) [84]. Indeed, we believe that the TLM\_CFM-F\_OSM combination's superior anti-cancer properties against NSCLC lung tumors are due to increased TGF-β and MMP-9 expression as well as increased CFM-F penetration into the tumors in this study.

STAT3 is one of the potential therapeutic targets for NSCLC. The level of constitutive STAT3 activation has been linked to lung cancer metastasis, angiogenesis, and resistance to a variety of anti-cancer drugs [85]. The chemotherapeutic sensitivity of OSM against non-small cell lung cancer cells was increased when STAT3 was suppressed by chemically modified siRNAs. STAT3 and NF-κB activation and interaction are crucial in controlling cancer cell-inflammatory cell communication. NF-κB and STAT3 are critical regulators of tumor angiogenesis and invasiveness in pre-neoplastic and malignant lung cancer cells [86]. We noted a significant reduction in STAT3 expression in H1975 tumors post-treatment with the TLM\_CFM-F\_OSM combination, which is consistent with these reports. Based on these findings, we hypothesize that NF-κB inhibition by the TLM\_CFM-F\_OSM combination regulates STAT3 expression and thus tumor growth and metastasis in H1975 tumors. Collectively TLM\_CFM-F\_OSM affects multiple pathways, including the AMPK, NF-κB, Lamin B2, and JAK-STAT pathways, as shown in Figure 6.

EVs from cancer cells contain microRNA, long non-coding RNA, small interfering RNA, DNA, protein, and lipids, which are all being studied for use in cancer diagnosis and treatment [87]. Shimada Y et al. investigated serum exosomal PD-L1 as a quantitative marker for predicting anti-PD-1 response and evaluating clinical outcomes in NSCLC patients [88]. In line with this study, EV markers from NSCLC tumors and serum showed significantly lower levels of exosomal markers (CD63, CD81, EGFR, and PD-L1) after treatment with the TLM\_CFM-F\_OSM combination in the present study. According to a growing body of evidence, EVs derived from NSCLC tumors also increased PD-L1 expression and, thus, tumor development, decreased CD8+ T-cell function, and induced CD8+ T cell death [89]. Furthermore, exosomal wild-type EGFR has been shown to cause OSM resistance in NSCLC (H1975) cancers [90], and exosomal EGFR was downregulated in this study by TLM\_CFM-F\_OSM treatment, suggesting that combination treatment affecting exosomal PD-L1 and EGFR expression could be helpful in reversing NSCLC tumor growth and OSM resistance.

NF-κB, Lamin B2, and JAK-STAT pathways, as shown in Figure 6.

MMP-9 inhibition, resulting in more nanoparticle penetration into the tumor [28,31,32]. As a result, TLM, in combination with any other anti-cancer drug, would be more effective in treating metastatic lung cancers. In addition, MMP-9 is involved in lung cancer invasion, metastasis, angiogenesis, and progression [83]. It negatively affects cancer immune modulation via TGF-β activation and intercellular adhesion molecule-1 shedding (ICAM-1) [84]. Indeed, we believe that the TLM\_CFM-F\_OSM combination's superior anti-cancer properties against NSCLC lung tumors are due to increased TGF-β and MMP-9 expres-

STAT3 is one of the potential therapeutic targets for NSCLC. The level of constitutive STAT3 activation has been linked to lung cancer metastasis, angiogenesis, and resistance to a variety of anti-cancer drugs [85]. The chemotherapeutic sensitivity of OSM against non-small cell lung cancer cells was increased when STAT3 was suppressed by chemically modified siRNAs. STAT3 and NF-κB activation and interaction are crucial in controlling cancer cell-inflammatory cell communication. NF-κB and STAT3 are critical regulators of tumor angiogenesis and invasiveness in pre-neoplastic and malignant lung cancer cells [86]. We noted a significant reduction in STAT3 expression in H1975 tumors post-treatment with the TLM\_CFM-F\_OSM combination, which is consistent with these reports. Based on these findings, we hypothesize that NF-κB inhibition by the TLM\_CFM-F\_OSM combination regulates STAT3 expression and thus tumor growth and metastasis in H1975 tumors. Collectively TLM\_CFM-F\_OSM affects multiple pathways, including the AMPK,

sion as well as increased CFM-F penetration into the tumors in this study.

**Figure 6.** Plausible mechanism of action of CFM4.17, Telmisartan and Osimertinib combination against non-small cell lung cancers in athymic nude mice. Telmisartan activates the PPAR-γ nuclear **Figure 6.** Plausible mechanism of action of CFM4.17, Telmisartan and Osimertinib combination against non-small cell lung cancers in athymic nude mice. Telmisartan activates the PPAR-γ nuclear receptor, CFM4.17 acts on CARP-1, and Osimertinib inhibits the EGFR mutated gene, increasing AMPK activity and thus regulating the p38 MAPkinase pathway, Lamin B2 protein, JAK-STAT pathway, PDL-1, and NF-κB pathway to maintain apoptosis, cancer metastasis, and immune suppression. Bcl-2: B-cell lymphoma 2, Bax: BCL2-associated X protein, CARP-1: Cell cycle and apoptosis regulatory protein 1, EGFR: epidermal growth factor receptor, CFM-F: lipid formulation of CFM4.17, MMP-1: Matrix metalloproteinase-1, OSM: Osimertinib, PD-L1: Programmed cell death 1 ligand 1, STAT3: Signal transducer and activator of transcription 3, SOX2: SRY-Box Transcription Factor 2, TLM: Telmisartan.

#### **5. Conclusions**

TLM\_CFM-F\_OSM showed a significant anti-cancer effect against H1975 tumor xenografts in athymic nude mice. Further, our in vivo studies with H1975 lung cancer cells demonstrated that this combination is effective through multiple pathways, including AMPK, NF-κB, Lamin B2, and JAK-STAT, which regulates mitochondrial homeostasis, inflammation, oxidative stress, and apoptosis. One novel mechanism of this triple combination in reducing the tumor burden of H1975 xenografts was the effect on serum exosome production and PDL1 and EGFR expressions. In addition, extensive molecular research is required to identify the specific molecular targets of these anti-cancer drugs for lung cancer treatment.

**Author Contributions:** R.N. and A.K.K. designed, performed, and analyzed the results and wrote the manuscript. N.P., S.K.S., L.S., A.B., N.K., E.N., R.S., P.A. and A.N. performed part of the experimental work and/or analyzed the results. D.G.M.J. performed TotalSeq assay from mouse serum EVs. M.S. conceptualized the study, wrote, and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** National Institute on Minority Health and Health disparities (National Institutes of Health), Grant/Award Number: U54 MD007582 and NSF-CREST Center for Complex Materials Design for Multidimensional Additive Processing (CoManD), Grant/Award Number: 1735968 for providing the funding for this research work.

**Institutional Review Board Statement:** The animal study was conducted according to the guidelines of the NIH, and approved by the Institutional Animal Use and Care Committee of Florida Agricultural and Mechanical University (**020-07** and 10 December 2021).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data that support the findings of this study are available on request from the corresponding author.

**Acknowledgments:** We thank the Florida A&M University and Florida State University for providing all kind of support, resources and excellent infrastructure to carry out this research work.

**Conflicts of Interest:** The authors declare that there are no conflict of interest.

#### **References**

