Nullomer peptide increases immune cell infiltration and reduces tumor metabolism in triple negative breast cancer mouse model

Background Nullomers are the shortest strings of absent amino acid (aa) sequences in a species or group of species. Primes are those nullomers that have not been detected in the genome of any species. 9S1R is a 5-aa peptide derived from a prime sequence that is tagged with 5 arginine aa, used to treat triple negative breast cancer (TNBC) in an in vivo TNBC mouse model. 9S1R is administered in trehalose (9S1R-NulloPT), which enhances solubility and exhibits some independent effects against tumor growth and is thus an important component in the drug preparation. Method We examined the effect of 9S1R-NulloPT on tumor growth, metabolism, metastatic burden, necrosis, tumor immune microenvironment, and the transcriptome of aggressive mouse TNBC tumors. Results The peptide-treated mice had smaller tumors in the initial phase of the treatment, as compared to the untreated control, and reduced in vivo bioluminescence at later stages, which is indicative of metabolically inactive tumors. A decrease in ex vivo bioluminescence was also observed in the excised tumors of treated mice, but not in the secondary metastasis in the lungs. The treatment also caused changes in tumor immune microenvironment with increased infiltration of immune cells and margin inflammation. The treatment upregulated 365 genes and downregulated 710 genes in tumors compared to the untreated group. Consistent with in vitro findings in breast cancer cell lines, downregulated genes in the treated TNBC tumors include Cellular Metabolic Process Related genes (179), specifically mitochondrial genes associated with TCA cycle/oxidative phosphorylation (44), and translation machinery/ribosome biogenesis genes (45). Among upregulated genes, the Developmental Pathway (13), ECM Organization (12) and Focal Adhesion Related Pathways (7) were noteworthy. We also present data from a pilot study using a bilateral BC mouse model, which supports our findings. Conclusion In conclusion, although 9S1R-NulloPT was moderate at reducing the tumor volume, it altered the tumor immune microenvironment as well as the tumor transcriptome, rendering tumors metabolically less active by downregulating the mitochondrial function and ribosome biogenesis. This corroborates previously published in vitro findings.

destroy mission, thus nullomer-based drugs may increase immune responses. The original peptoprime sequences described in 2007 [14] are ve amino acids long and have been modi ed with the addition of ve arginine molecules to enhance cell-penetrating properties [17,18]. When dissolved in trehalose, several of the peptoprimes (NulloPTs) were shown to be preferentially lethal to breast and prostate cancer cells, as opposed to normal primary cell lines [17][18][19]. Trehalose is reported to have an independent effect against BC [20][21][22][23][24] and is an important component in our drug preparation. Nullomers have also been used to design vaccines from epitopes present in the host but absent in a pathogen and to make DNA "watermarks" for labeling by combining them in series [19].
The original 198 peptoprimes (and scrambled versions of their sequences) were assayed in vitro against the NCI 60 cancer lines [18], and 9S1R, a potent killer of cancer cells was selected for the mouse experiments reported here. 9S1R is a scrambled version of an original peptoprime, that is a nullomer (absent) from the biome. 9S1R-NulloPT is absent from the human and mouse proteomes and has been shown to dramatically decrease ATP production, inhibit mitochondrial F o F 1 -ATP synthase, reduce mitochondrial membrane potential and increase superoxide free radicals inside the mitochondria of MDA-MB-231 human TNBC cells and the NCI 60 cancer cell panel, ultimately killing them [17,18] This is the rst in vivo study of nullomer-based peptides. Here we report the therapeutic effects of 9S1R-NulloPT in the 4T1.2-Luc mouse TNBC model. The 9S1R-NulloPT was initially tested in a bilateral tumor model (pilot study-1, details in Supplementary) with six administrations at two different doses (50mg/kg and 100mg/kg). Following these results, we moved to a unilateral tumor model with eight drug administrations at the most effective dose (100mg/kg). Although our preclinical in vitro studies showed a substantial response against diverse panels of cancer cells, the present in vivo study in mice showed that the drug effectively reduces TNBC tumor size at the initial growing phase of the tumor, but not in the advanced stage. 9S1R-NulloPT changes the tumor immune microenvironment and affects tumor energy metabolism speci cally targeting mitochondrial and ribosomal genes, providing a segue for future studies targeting tumor mitochondria and ribosome synthesis in TNBC.

Methods
1. Drug preparation-9S1R nullomer peptide (sequence RRRRRWCMNW) was synthesized by Genscript (USA) (lyophilyzed, HPLC puri ed, purity > 98%) and stored at − 20 • C. For preparation of 9S1R-NulloPT, brie y, 20 µg/µl stock solution of the 9S1R peptide was prepared in 100mM Trehalose (Sigma-Aldrich, USA), which was then used as mg/kg body weight for injections in mice. For example, a mouse of 25g body weight for dose 100mg/kg, received 200ul of drug formulation containing 100ug/g bodyweight of 9S1R peptide mixed with 2.5mM/g bodyweight of Trehalose prepared in PBS. Trehalose only group received 200ul of 2.5mM trehalose in PBS per gram bodyweight, and PBS group received 200ul of PBS. All drug preparations were ltered through 0.22µm lter (Millipore, USA) before IP administration.
2. Single-dose acute toxicity study-A single dosage of the 9S1R-NulloPT drug at 5, 25, 50, 100 mg/Kg and highest equivalent dose of Trehalose alone (2.5mM/g bodyweight) was injected in mice (n = 4) intraperitoneally (IP). Mice were observed every 2h for clinical signs and symptoms and body weights were taken every 12h. At the end of the experiment on 36h mice were euthanized, necropsy was performed and organs preserved in 10% formalin solution.
3. Cell culture: Mouse triple-negative 4T1.2 and 4T1.2-Luc cells were cultured and maintained as previously described [25] with α-MEM supplemented with 10% fetal clone III serum (Cytiva, USA), 1% penicillin/streptomycin and 1mM sodium pyruvate. Cells were maintained at 37 C, 5% carbon dioxide, and 100% humidity in a sterile tissue culture incubator. 4. The TNBC mouse model and treatment paradigm-The model was created by orthotopically injecting gene count for each sample was done using median-ratio-normalization. Volcano plots, PCA plots, heatmap of samples (using Euclidean distances), heatmap of expression of top variable genes were generated for using gplots, ggplot2 and RColorBrewer programs (R based). Fold-change for each gene was calculated by comparing counts in all the treatments relative to vehicles and PBS. Genes with Benjamini-Hochberg (BH) adjusted p-value < 0.05 were considered differentially expressed genes (DEGs).
Differentially expressed genes having log2 fold change value of at least 0.58 or greater were used for investigating signi cant biological processes, molecular functions, cellular compartments and biological pathways using gPro ler web server.
11. Interactome and cluster analysis-To explore if the DEGs are involved in known and predicted proteinprotein interactions, the TNBC-related PPI network was constructed using the online analysis tool STRING (Ver 11.2). The network nodes were the downregulated and upregulated DEGs and network edges shown in con dence view without any disconnected nodes in the network and with active interaction sources from experiment, databases, co-expression, neighborhood, gene fusion and co-occurrence [26]. The network was clustered to 3 groups following kmeans clustering with hidden edges between clusters for a simpli ed view. The interaction score was > 0.4 [27].
12. Statistical Analysis-Two-tailed unpaired Student's t-test was used for experimental statistics with p < 0.05 considered as statistically signi cant. Students' T test and one-way ANOVA followed by post hoc test. GraphPad Prism (ver 9.5.1) was used for analysis.

Results
Characterization of the peptide 9S1R 9S1R (N-RRRRR-WCMNW-C) nullomer has a molecular weight 1519.81 Da and iso-electric point pH 12.28.
It has a net charge of + 5 at pH 7, hydrophobicity + 12.93 Kcal/mol with amphipathic nature and fair solubility in water (Fig. 1A, B). We evaluated the uptake of the peptide inside 4T1.2 cells by mass spectrometry (MS) and found that the peptide is available inside the cells at two molecular forms with charges 3+ (m/z 507.2637 Da) and 4+ (m/z 380.7002 Da), (extracted ion chromatogram (EIC) peaks, Supplementary Fig. 1). We determined using MS that the half-life of the peptide in fetal bovine serum (FBS) was 8.7 mins (Fig. 1C). The previous in vitro study by Alileche et al. showed that this peptide targets mitochondrial function [18]. This is in agreement with the above properties of this molecule, in that it is very similar to known mitochondria-targeting peptides (MTPs) [28-31] and, we hypothesize that the peptide localizes to mitochondria as an MTP. Thus, we determined that the peptide enters cells and has a short half-life in serum.
Single dose acute toxicity study of 9S1R NulloPT in mice.
To determine safety of our peptide formulation in vivo, we performed a single dose toxicity study in female BALB/c mice ( Fig. 2A). The drug doses (administered I.P.) used are 5, 25, 50 and 100 mg/kg, with the control being the highest trehalose dose volume. After 36 hours of observation the experiment was terminated, and we found no signi cant change in the animal behavior, clinically observable signs of acute toxicity or conspicuous change in body weights among any groups (Fig. 2B). There was no signi cant difference between the drug and trehalose alone group. All the drug doses were well tolerated by the animals, allowing us to move forward with the preclinical in vivo cancer studies.
Treatment with 9S1R-NulloPT inhibits tumor growth in early stage cancer but does not affect tumor volume in late stage growth.
We evaluated the effect of 9S1R-NulloPT on female BALB/c mice (8 weeks old), using a syngeneic 4T1.2-Luc orthotropic model of metastatic TNBC. We investigated the effect of the drug on tumor growth over one month in both a bilateral model (initial pilot study, Supplementary Fig. 2.) and a unilateral model ( Fig. 3A and Supplementary Fig. 3). In both studies, there was a drop in weight following the rst week's treatment, however the mice later returned to near-control level weight (Fig. 3B, Supplementary Fig. 2C). The weight loss was never greater than 15% of the initial body weight, ruling out any adverse effects. We found that although the 9S1R-NulloPT treated mice maintained a reduced tumor volume as compared to untreated controls (

9S1R-NulloPT alters tumor metabolism but not metastasis
Monitoring bioluminescence of the tumor cells in vivo revealed that, although the initial tumors (day 8) had similar signals in both control and treatment group, the control group tumors displayed increased in vivo bioluminescence with time. Five of 8 treated animals showed a reduction at the later time points (day 22 and day 28) (Fig. 4A, B). We found that the excised tumor weights among the groups were not signi cantly different at the end of the study (Fig. 3); however, the ex vivo BLI of the excised tumors (post mortem) showed a signi cant decrease in signal with treatment ( Fig. 4C, D). This suggests that tumors from the treatment group have a decreased number of metabolically active 4T1.2-Luc cells. Fire y luciferase oxidizes luciferin by using ATP, Mg 2 + and O 2 [33], and depletion of cellular ATP results in lower luminescence along with loss of metabolic functioning. It has been previously shown in vitro that 9S1R-NulloPT completely depletes the cellular ATP of BC cell lines within 3h and are highly lethal to hormone independent and triple negative BC cell lines (MDA-MB-231, BT-549, HS-578 T, and MDA-MB-468), as well as hormone dependent BC cell lines (MCF-7 and T-47D) [17,18].
Although there was a trend of reduction, there was no signi cant change in the bioluminescence of cells metastasizing to lung (Fig. 4E, F) and no change was seen in the number of metastasis to lungs after treatment (Fig. 4G). These ndings corroborated our previous study (Supplementary Fig. 2A while none were found in the control groups. The results indicate that 9S1R-NulloPT treatment alters the TME and enhances the immune and in ammatory response. This is corroborated by the increased number of plasma cells [34], tumor-in ltrating lymphocytes (TILs) [35,36] and higher immune scoring [37], which are all associated with a positive prognosis in TNBC patients.
Interestingly the Igkv (immunoglobulin kappa variable) family (Igkv 12-44, Igkv 3-4, Igkv 4-58, Igkv 6-15, Igkv 6-23 and Ighe) were among the top 10 downregulated genes (Fig. 6A). We investigated the presence of clusters in the interactome network of the differentially expressed genes (DEGs) from total upregulated and downregulated genes, using STRING software (Ver 11.5) [27]. Of the upregulated DEGs, we found clusters speci c to cancer, focal adhesion, signal transduction, cell adhesion and nervous system development (Fig. 6C). Network analysis of downregulated genes revealed an overall cluster for metabolic process related genes with speci c clusters for mitochondria, ribosome and translation machinery, immune system and myo bril assembly (Fig. 6D). Functional enrichment analysis using the ve highest signi cant gene ontology (GO)-terms (biological process, molecular function and cellular component) and pathways (from Kegg, Reactome and Wikipathways) of upregulated and downregulated DEGs con rmed the results [38,39]. The details of the enrichment analysis and list of pathways are provided in Table 1. 9S1R-NulloPT speci cally targets metabolic and bioenergetics pathways.
We found cancer related pathways, and at least 65 cancer pathway-associated DEGs, were upregulated with treatment. Since, we compared treated TNBC tumors with untreated TNBC tumors we investigated whether such cancer related pathways and DEGs are contributions of the cancer model or an effect of drug-induced change. To achieve this, we used publicly available RNA-Seq data by Schrors et al [40], comparing gene expression of 4T1.2-Luc TNBC cells (derived from BALB/c mammary gland) versus normal BAlb/C mammary tissue. This comparison identi es the DEGs that result from BC (TNBC) alone. Our 4T1.2-Luc cell line, developed by Jorcyk et al. [25], is a luciferase tagged single cell clone of 4T1.2 cells [41] with similar genomic background and tumor properties to the original 4T1, but with a higher tendency to metastasize. A comparison of the DEGs from 9S1R-NulloPT treated vs untreated tumors (group a), and 4T1 tumor cells vs mammary cells (group b) identi ed those DEGs that resulted from 9S1R-NulloPT treatment (unique from group a). DEGs resulting from TNBC [42,43] are common to group a and b (Fig. 7).
The comparison between the RNA-Seq results revealed that 688 DEGs were due to TNBC (common to group a and b), with 233 genes upregulated and 455 genes downregulated (Fig. 7A). Cluster analysis of these genes using STRING (Ver 11.5) revealed the TNBC downregulated clusters consist of myo bril assembly, immune related and mitochondrial electron transport chain (ETC)TC related genes. The TNBC upregulated cluster comprises focal adhesion and cancer signaling related genes (Fig. 7C). Although no noteworthy cluster was found in upregulated genes unique to 9S1R-NulloPT treated cancer, Abl-1 and Shc-1 formed the center node of a small cluster. Remarkably, when analyzed in details ( Supplementary  Fig. 5), among the above-mentioned cancer related pathways we found only 20 associated genes to be uniquely upregulated in 9S1R-NulloPT treated tumors ( Supplementary Fig. 5A-C) including pan-cancer related genes such as Shc-1 [44], Mtor [45], Lrp6 and Wnt5a [46]. Interestingly, a recent report suggests that Wnt5a [47] and Abl-1 [48] are potent suppressors of TNBC progression and are associated with a better prognosis in BC. We also found 44 DEGs associated with at least 12 cancer pathways that were upregulated in both 9S1R-NulloPT treated and TNBC untreated tumors ( Supplementary Fig. 5, D-F), signifying a contribution from the TNBC model. A list of cancer pathway related DEGs in TNBC untreated tumors and 9S1R-NulloPT treated tumors is provided in Supplementary Fig. 6.
The unique downregulated clusters, which signify the effect of 9S1R-NulloPT only (and not breast cancer), comprise mitochondrial ATP synthesis coupled proton transport (Atp5e, Atp5j, Atp5k, Atp5h and Atp5j2), mitochondrial electron transport chain (Uqcrb, Uqcrh, Ndufv2 and Ndufs6), Oxidative phosphorylation (Cox6c, Atp5e, Uqcrb, Ndufs6, Atp5j, Cox7a2, Atp5k, Uqcrh, Atp5h, Ndufv2 and Atp5j2), Mitochondrial respiratory chain complex-1(Ndufv2, Ndufs6, Ndufs5, Ndufa5, Ndufb5, Ndufs4 and Ndufb3) and ribosome/translation related genes (Rpl17, Rpl19, Rps27l, Rpl22l1, Rpl9, Rps14, and Rps27) (Fig. 7B, E, F). The top 5 unique downregulated genes were from the Igkv family (Igkv 3-4, Igkv-12-44, Igkv 4058, Ighe and Igkv6-23) and top 5 unique upregulated genes were Adgrl, Mgat3, Lhx6, Map1a and Spef1 (Fig. 7D). Enrichment analysis of the mitochondria cluster revealed Cristae formation, Complex I biogenesis, Oxidative phosphorylation, Electron transport chain, TCA cycle pathways and the GO-terms such as respirasome, mitochondrial respiratory chain assembly and mitochondrion organization (Fig. 7E). Enrichment analysis of the ribosomal cluster revealed Ribonucleoprotein complex, RNA binding, Ribosomal biogenesis, Translation and Peptide biosynthesis pathways and the GO-terms such as rRNA processing, SRP dependent cotranslational protein targeting to membrane, L13a mediated silencing of Ceruloplasmin expression (Fig. 7F). Interestingly, low ceruloplasmin expression correlates with a favorable prognosis and tumor immune cell in ltration in BC patients [49]. We found 149 genes which were upregulated with TNBC (in group-b) were downregulated as an effect of 9S1R-NulloPT treatment (in group-a). These genes comprise the same cluster of Ribosome and Mitochondria genes with similar GOterms and pathways ( Supplementary Fig. 6A-C). Likewise, 99 genes which were downregulated as an effect of TNBC (group-b) were upregulated due to 9S1R-NulloPT treatment (group-a), and these includes Focal adhesion, nervous system development, ECM organization related genes ( Supplementary Fig. 6D-F). In summary, the RNAseq suggest that 9S1R-NulloPT targets metabolic pathway related genes, speci cally the Mitochondrial energy metabolism and Ribosome associated pathways such as Ribosome biogenesis and translation.  [17,18] where it is reported that 9S1R-NulloPT reduces ATP formation and mitochondrial membrane potential, and increases mitochondrial reactive oxygen species (ROS) generation. A moderate to mild mitochondrial stress and ssion is bene cial for TNBC cell growth and aggressiveness, but severe mitochondrial stress and increased ssion leads to excessive ROS generation and apoptosis of cancer cells [50]. The drastic alteration in the mitochondrial pathways in the 9S1R-NulloPT treated tumors shows that the peptides target mitochondria directly or indirectly, which could be exploited as a potential mitochondria-targeted anti-cancer/tumor therapy. Ribosome synthesis in the nucleolus increases in cancer cells to cope with increased demand for protein synthesis. Ribosome biogenesis-targeting (CX-3543, CX-5461 [51,52] and BMH [53]) is still in its infancy, however, it is emerging as an effective cancer therapy used in multiple clinical trials [54]. Biogenesis of ribosomes make an interesting target for cancer chemotherapy for many reasons: (1) the inhibition of ribosome biogenesis induces cell cycle arrest in a p53-independent manner [54], (2) these inhibitions don't affect the resting cells, possibly due to the long half-life of cytoplasmic ribosomes [55], and (3) it could lead to apoptosis of neoplastic cells that have a high nucleolar ribosomal biogenesis rate [56]. Ribosome synthesis is extremely complex and one of the most energetically demanding cellular activities [57]. We hypothesize that our drug 9S1R-NulloPT targets mitochondrial ATP production and ribosome biogenesis leading to a loss of metabolic activity of the cells. To ensure more e cient killing of these aggressive TNBC cells in vivo and to enable tumor size reduction, we plan to use our drug synergistically along with other anticancer drugs that act through different cytotoxic pathways [58-61].
RNAseq analysis revealed upregulation of: unique clusters of ECM organization genes (collagen formation and degradation genes such as Col6a3, Col5a3 Plod1, Mmp3, Loxl3 and Plec); focal adhesion genes (Itga5, Lamc1, shc1, Arghap35, and Pxn), and cytoskeletal protein binding genes (Abl1, Map1a, Map1b, Map6, Myo10 and Trak1). Among the highest upregulated genes Lhx6 was noteworthy in relevance to BC, as it is reported to suppress activation of the PI3K/Akt/mTOR signaling, inhibiting the progression of BC [62]. The highest downregulated genes belonged to the Igkv family, which has been suggested as an identifying biomarker for TNBC cancers [63]; and the Fcmr gene whose knock down leads to increased phagocytosis, enhanced antigen presentation, and heightened T cell activation. Fcmr is also a promising anti-cancer target [64].
Although treatment did not change tumor size post necropsy, the in vivo as well as ex vivo BLI of the tumors showed reduced bioluminescence in the treated group. There were multiple mice (~ 50%) in the treatment group that showed a reduction in bioluminescence which implies less cells being present in the tumor due to decreased proliferation or an increased cell death and loss of metabolic activity of the tumor. This corroborates the results obtained in the pilot study. Ex vivo BLI of the tumors in both the studies showed a signi cant decrease in bioluminescence and thus a decrease in the metabolic activity of the tumor cells in the treated groups but not in controls. This suggests that although the treatment does not change the size of tumors drastically, it altered the metabolism of the tumor cells rendering them inactive. This makes sense in light of the tumor transcriptomics: metabolism related genes were mostly downregulated in the treated tumors, speci cally the mitochondrial and ribosomal genes that are essential for energy production and metabolism. The in vivo BLI from day 22 (24h after dose 5) clearly shows the reduced bioluminescence in treated tumors during this period. For the pilot study (using a bilateral tumor model) we imaged mice after the 4th 9S1R-NulloPT dose (day 22, Supplementary Fig. 4A) and found a similar decrease in in vivo bioluminescence in the treated groups.
Histopathological evaluation con rmed that treatment and control tumors had the same grade, stage and aggressiveness. However, it was interesting to nd that there was a signi cant change in the tumor immune-microenvironment of the treated tumors. Immune cell in ltration increased signi cantly in the 9S1R-NulloPT treated tumors, as measured by plasma cell numbers and margin in ammation. A positive correlation of plasma cells with favorable patient outcomes has recently been reported, [34] suggesting a better prognosis. It is well established that the outcome of immunotherapy treatment, and thus prognosis of BC, is dictated by the tumor microenvironment. Recent reports suggest that the immune score of BC patients could be useful for treatment decisions and survival prediction [37]: activated immune cell in ltration in tumors correlates with better prognosis [35]. Increased levels of tumor in ltrating lymphocytes (TILs) have been associated with disease-free status and overall survival rates in TNBC patients with and without any treatment. The presence of TILs in the breast tumor microenvironment can also predict responses to neoadjuvant therapy and adjuvant chemotherapy treatments, and high numbers of TILs correlate with increased pathological complete responses in TNBC [36]. We also found that the 9S1R-NulloPT treatment resulted in less externally visible necrosis as compared to untreated tumors or trehalose treated groups (data not shown). There was no conspicuous difference between the PBS control and trehalose groups (Supplementary Fig. 4A-G), suggesting that the immunological changes in the tumor microenvironment (TME) are a speci c contribution of the nullomer peptide.
The results of this study should be seen in the light of the fact that the peptides were not stabilized and have a half-life in serum of about 9 minutes. Small peptides such as 9S1R tend to have shorter half-life in circulation, but when internalized their response could be effective immediately as we have seen in MDA-MB-231 cell lines [18]. Cellular internalization studies ( Supplementary Fig. 1) show that the peptide could be identi ed from crude lysed cytosol fraction (cytosol + mitochondria fraction) in several valence forms (2+, 3+, 4+) from m/z 380.7 0.1 when incubated with 4T1.2 cells for at least two hours. This con rms that the 9S1R-NulloPT are internalized within the cells and are sequestered into other forms, which are more stable than those in serum. Biochemical properties of 9S1R are similar to known mitochondria penetrating peptides in charge, hydrophobicity, poly arginine content [65], and the effects on mitochondrial functions [17]. All of this suggests mitochondrial localization. However, the mechanism of the peptide's metabolism, uptake and its sub-cellular localization warrants further investigation.
The Nullomer peptides are readily solubilized in the sugar trehalose, which has known anti-cancer properties [23,66]. We used the nullomer-trehalose combination with the peptoprime 9R-and its scrambled version 9S1R to screen the NCI 60 cancer panel and the TNBC model MDA-MB-231 and found them to be effective in vitro. Another peptoprime linked to 5 arginines, did not show anticancer effects, and is used as a negative control peptoprime [17,18]. As noted above, trehalose is reported to exhibit anticancer properties, and in this study, we did see some effects ( Supplementary Fig. 2-5). Trehalose alone had similar potency in tumor size reduction as 9S1R-NulloPT groups in study-2 ( Supplementary Fig. 3), but a contrasting higher than control tumor size in study-1 ( Supplementary Fig. 2), with no signi cant change in post mortem tumor size. Similar to the control group, Trehalose did not show any effect on the immune cell in ltration or margin in ammation on the tumors (Supplementary Fig. 4).
The 4T1.2-Luc cells have a speci c tendency to metastasize into lungs and bone. We found speci c metastasis to the lungs, and although there was a trend of reduced bioluminescence in the treated lungs as compared to controls, it was not statistically signi cant, this is corroborated in the pilot study. There was also no signi cant change in metastatic foci counts in the lungs. These metastatic ndings are still preliminary, primarily because of the large variations in the control groups.
Overall, we show a moderate level of therapeutic potential of 9S1R-NulloPT, in terms of tumor growth. In (2) The RNA sequencing results revealed mitochondrial and ribosomal genes to be greatly affected by 9S1R-NulloPT treatment, but this needs to be con rmed by PCR. We also plan to identify the targets directly by the subcellular localization of the 9S1R-NulloPT.
(3) To improve the therapeutic effect of the drug we plan to combine it with a known chemotherapy drug such as Doxorubicin along with 9S1R-NulloPT in in vivo TNBC models.
(5) RNA seq analysis should be repeated with mammary tissue from normal mice, those with TNBC tumors, and those with TNBC tumors treated with 9S1R-NulloPT, in at least two different stages (day 18 and day 27).
(6) We did not perform any intravenous administration as we found previously that 9S1R-NulloPT has a low but signi cant hemolytic activity of 0.5% at 10 µM concentration on RBCs [17].

Conclusion
Previously in vitro experiments with the NulloPTs showed very promising results using the NCI 60 panel of cells. he current in vivo mouse study (Fig 8) demonstrates that the drug reduces early tumor volume    harmonizing the data from both current and pilot studies, data aligned by injection timepoints. The green box depicts the responsive window with a statistically signi cant difference between the control and treated groups by the 4th dose (p<0.01, n=10-11). Data presented as Mean+ SEM followed by two-tailed unpaired Student's t-test, *p<0.05 considered statistically signi cant.    Comparative analysis of the effect of cancer vs effect of 9S1R-NulloPT treatment, in mice mammary tissue: DEGs from a published study [40]