StemCell Therapy

One of the major hurdles in cancer therapy is the lack of appropriate treatments that would annihilate the cancer cells to increase the survival of patients. Thus, combinations of various chemicals were used based on the overall molecular phenotype of the type of cancer cells. Therefore, any new strategy that would help in the treatment of any aggressive form of cancer will be welcoming. One such aggressive form of cancer is TNBCs. Current treatment option for treating TNBCs cantered around paclitaxel6,7 and neo-adjuvant therapy8,9. Over a decade, it is becoming clear that stem cell conditioned medium containing secretome may be a good therapeutic in treating cancer35,36,37,38,39,40,41,42,43,44,45. Thus, we here show the functional and biochemical characterization of MSC secretome and its effectiveness in inducing apoptosis in TNBC cells.

Though previous studies had characterised and identified proteins52,53,54 in the secretome, we have prepared the stem cell secretome in a GMP compliant55 FDA approved stem cell media and quantified all the major biomolecules in the freeze dried powder (Fig.1). On the same note, lipid spectral characteristics seen in Fig.1h is indicative of anti-cancerous nature as suggested by Brossa et al.56. Mineral components profiled in Fig.1 could be the crystalline particles seen in control (Fig.1a) and flakes could be formed from protein (growth factor supplements in the chemically defined medium of stem cells) FESEM image (Fig.1a) and (carbohydrate) glucose quantified in Fig.1f. Ca, P and Fe are essential elements constituted as inorganic nutrient supplements in such media. Fe is a key element in cancer cell metabolism and Ca and P are thought to play a beneficial role in cancer. These minerals present in secretome and not present in the form of crystals on flakes in Fig.1b could be due to the presence of metallo-proteins in secretome. Protein and RNA are major components in freeze dried secretome and could be forming the needle like structures in Fig.1b. Exosomes57 present in the secretome could not be definitively identified in control, Fig.1a.

Although stem cell secretome was prepared by many by conditioning stem cells in a growth medium for a specific time35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,52,53,54, the process if not controlled52 and balanced with respect to incubation conditions could lead to nutrient deprivation resulting in cell death or growth arrest35,36,37,38,39,40,41,42,43,44,45. As a result, contradicting results have been reported46,47,48,49,50 and could be attributed to the variability in preparation and processing methods. Thus, to address this issue, we designed our first experiment to address the issue of whether the anti-cancer effects reported35,36,37,38,39,40,41,42,43,44,45 was the outcome of using stem cell conditioned media that is deprived of nutrients or due to the secretome. Towards this, freeze dried secretome were supplemented with nutrients in chemically defined media of TNBCs (CDM). The media alone significantly increased population of cancer stem cells (CD44+/CD24 phenotype) in TNBCs (Fig 2a) likely due to the presence of insulin, hydrocortisone and epidermal growth factor that are known to contribute to colony formation characteristic feature of cancer stem cells51. However, the same markers were substantially decreased when the same media contained secretome (Fig. 2g). For the first time, we show a dose dependent cytotoxic effect of SF which is similar to the anti-cancer effect of secretome reported previously35,36,37,38,39,40,41,42,43,44,45. At 20 mg/ml, the effect on cancer cell death was 50% (Fig. 2b). However, at higher concentrations of SF significant cancer cell death could be observed (Fig. 2c) up to 80%.

It is clear from Fig.2e with different amounts of lyophilized powder of the chemically defined media of MSCs added to the chemical defined media of TNBCs without exposure to MSC cells (secretome free) that the cell viability shown in Fig.2e was not affected compared to control. This suggested there was no effect of changes in the salt concentrations in the chemically defined medium affecting cellular homeostasis. Further, SF dose dependent decrease in cell viability (Fig.2c) corresponded with in Fig.2f showing cellular apoptosis, which suggested that decreased cell viability observed, is indeed due to the result of apoptosis. Since the cell line we used belonged to mesenchymal stem like and mesenchymal like category (MDA-MB-231) among the six different TNBCs due to heterogeneity, we checked for stemness markers CD44+/CD24 and MDR1+4,5 (multi drug resistance protein 1), and also PDL1+6,58 (Programmed Death Ligand 1) as these population of cells can be potentially harmful with respect to invasion and aggressiveness. To our surprise, the percentage of total CD44+/CD24, MDR1+ and PD-L1+ TNBCs following treatment with SF was brought down dramatically (Fig.2g) suggesting the significance of secretome medium in the removal of these aggressive phenotypes from the TNBC population. To further verify our findings we checked the effect of SF on xenograft models of TNBCs.

Tumor microenvironment is known to play a critical role in the response to therapy59 and hence to confirm the anti-cancer potential, tumor spheroids were treated with SF. Results in Fig.3a and supplementary Fig.S2B showed that IC50 value of SF in 3D culture was 32.57mg/ml three times of the IC50 value in 2D culture shown in Fig.2c and Supplementary Fig.S2A, the latter being only 10.54mg/ml SF. Thus there was found evidence indicating greater resistance of spheroids to secretome formulation treatment. It was interesting to note that 50mg/ml SF in 3D culture (Fig.3a) that had similar effect to 20mg/ml SF in 2D (Fig.2c) signifying a difference in response associated with more stem like characteristics59 and metabolic deregulation60.

SF was administered into the tumor for 21 consecutive days in the light of treatment duration previously reported by others10,11,12,41. Thus, the calculated equivalent dose corresponding to once a day intra-tumor administration of 10X secretome is found to be 200mg/ml41. Thus, we treated tumor with 50 and 100mg/ml SF led to undetectable TNBCs from 1mm3 tumor stroma (Fig.3b). As shown in Fig.3b, c, significant suppression of overall tumor growth and CD44+/CD24, MDR1+3,4,5 and PD-L1+6,58 cell types that are involved in stemness and invasion was observed indicating the significance of SF in effectively controlling TNBC growth. Indeed, eradication of these populations has immense value in developing therapies for cancer as cancer stem cells very much drive tumorigenesis, metastasis and therapeutic resistance3,4,5,6,7. The histopathology data of Fig.3d clearly showed necrotic tumor patches with murine cells in the vicinity that developed after SF in vivo treatment.

Figure4a shows that there was no significant difference between 1nM and 100M compared to the reported 100% effective concentration of 10nM61. This is evidence of paclitaxel resistance. Interestingly, the extent of reduction of CD44+/CD24 breast cancer stem cells, multi drug resistance protein 1 (MDR1), programmed death ligand-1(PD-L1) expressing cells were far less than that due to SF seen in Fig.2g. It is to be noted that many reports indicate the use of taxane based therapies for TNBC type9 to overcome the drug resistance mechanisms associated with these cell types3,9. Thus, we compared the effect of paclitaxel with SF by treating TNBCs with SF and paclitaxel near to its IC50 value and beyond singly or in combination (Fig.4). Although, at the IC50 value of paclitaxel we observed significant cell death, a significant population of TNBCs were resistant to paclitaxel and that SF medium alone at 70mg induced significantly more death of TNBCs than paclitaxel (Fig.4ac).

It has been reported that tumor micro environment plays significant role in determining the therapeutic efficacy of paclitaxel3,59. Thus, we verified the effect of SF and paclitaxel in a 3D environment to find its influence on the observed cell death. Using the developed 3D cultures of TNBC (Fig.5), we found similar pattern of cell death as observed in our 2D and in vivo studies confirming the effect and significance of SF medium in inducing cell death. Nonetheless, Figs. 4c and 5 confirms prospective use of SF 70mg/ml in combination with lower doses of paclitaxel. This clearly demonstrate that a combination of MSC secretome with lower doses of paclitaxel can be used to minimise toxic side effects of using higher doses of paclitaxel as previously reported62 in the treatment of highly chemo resistant TNBCs in clinics.

All the results discussed in Figs.1, 2, 3, 4 and 5 suggest the prospective therapeutic potential of SF for clinical management of highly chemo resistant cancer types. Perhaps SF formulation could also favour efficacy of paclitaxel at lower dose which is otherwise highly neuro-toxic when administered at higher doses62. It would be interesting to fractionate or separate each of the biomolecule in Fig.1 to further study the contribution of them towards the anti-cancer effects discussed. In conclusion our study clearly illustrates that SF is effective in suppressing tumor growth and inducing TNBC cell apoptosis in a manner efficient than paclitaxel. Importantly, cells with PDL1+, CD44+/CD24, MDR1+ phenotypes that are potentially harmful with respect to invasion and aggressiveness could significantly be eliminated from TNBC population. Moreover, combination of SF with paclitaxel could be used to reduce the toxic effect of paclitaxel and add value to the finding. In addition, targeting the genes associated with cancer progression and survival following SF treatment could improve the treatment outcome. Further in-depth gene expression studies are required to apprehend the detailed molecular path ways associated with apoptotic mechanisms that are responsible for the anti-cancer potential of stem cell secretome. Protein and RNA characterisation of the secretome would also help to identify molecules responsible for nucleic acid degradation seen in spheroids and mechanisms involved in immune cell infiltration into tumor stroma. Based on our results, we envisage the potential of SF in combination therapies for the treatment of aggressive forms of TNBC.

Original post:
Adipose derived mesenchymal stem cell secretome formulation as a biotherapeutic to inhibit growth of drug resistant triple negative breast cancer |...

This entry was posted on December 8, 2021 at 1:52 am and is filed under Stem Cell Negative. You can follow any responses to this entry through the RSS 2.0 feed. Both comments and pings are currently closed. |