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Breast cancer-derived DAMPs enhance cell invasion and metastasis, while nucleic acid scavengers mitigate these effects

Breast cancer (BC) is the most common malignancy in women. Particular subtypes with aggressive behavior are major contributors to poor outcomes. Triple-negative breast cancer (TNBC) is difficult to treat, pro-inflammatory, and highly metastatic. We demonstrate that TNBC cells express TLR9 and are responsive to TLR9 ligands, and treatment of TNBC cells with chemotherapy increases the release of nucleic-acid-containing damage-associated molecular patterns (NA DAMPs) in cell culture. Such culture-derived and breast cancer patient-derived NA DAMPs increase TLR9 activation and TNBC cell invasion in vitro. Notably, treatment with the polyamidoamine dendrimer generation 3.0 (PAMAM-G3) behaved as a nucleic acid scavenger (NAS) and significantly mitigates such effects. In mice that develop spontaneous BC induced by polyoma middle T oncoprotein (MMTV-PyMT), treatment with PAMAM-G3 significantly reduces lung metastasis. Thus, NAS treatment mitigates cancer-induced inflammation and metastasis and represents a novel therapeutic approach for combating breast cancer.


INTRODUCTION
Breast cancer (BC) remains the most common malignancy in women worldwide and the second leading cause of cancer-related mortalities in the United States. 1,2 Metastatic disease accounts for over 90% of cancer-related mortality in breast cancer. [3][4][5][6] Once metastasis has developed, treatment options are limited, leading to a decrease in patient overall survival. Thus, developing strategies to block metastasis is urgently needed to improve patient outcomes. [5][6][7][8] Significant progress has been made in the development of targeted therapy for breast cancers that are estrogen/progesterone receptor positive (ER + /PR + ) and human epidermal growth factor receptor 2 positive (HER2 + ). Unfortunately, the current therapies are not as effective in the treatment of the most aggressive and metastatic-prone breast cancer subtype, termed triple-negative breast cancer (TNBC), which lacks the molecular targets ER, PR, and HER2. [9][10][11] TNBCs are seen with higher frequency in younger patients and tend to have higher rates of local recurrence and distant metastasis when compared to other breast cancer subtypes. 9,12 Standard of care for TNBC treatment includes a multi-agent chemotherapy regimen of taxane-and anthracyclinebased therapeutics (e.g., nab-paclitaxel and doxorubicin) along with surgery. [13][14][15] Despite recent advances in multimodality chemotherapy treatments, TNBC patient prognosis remains poor; for patients with advanced metastatic disease, there is a 3-year overall survival of 58% in TNBC patients compared to 80% in non-TNBC patients. 16,17 Thus, an unmet clinical need exists for developing improved TNBC therapies. 18,19 Originally designed for non-viral nucleic acid delivery, 20-23 our group identified a novel application for the cationic polymer polyamidoamine generation 3.0 (PAMAM-G3) as an extracellular nucleic acid scavenger (NAS). Since cell-free DNA (cfDNA) and other nucleicacid-containing damage-associated molecular patterns (NA DAMPs) have been implicated in aberrant toll-like receptor (TLR) signaling in several inflammatory diseases, [24][25][26][27][28] we and others have investigated the use of NASs in blocking pro-inflammatory signals through the binding of NA DAMPs in various disease models, including murine models of acute toxic shock syndrome, thrombosis, lupus, rheumatoid arthritis, sepsis, and pancreatic cancer (PC). [29][30][31][32][33][34][35][36] Tumor progression is associated with the production of numerous inflammatory mediators, including cytokines and chemokines, and the recruitment and activation and subsequent functional modulation of leukocytes, particularly mast cells, macrophages, and neutrophils. 37,38 Inflammatory mediators are sensed by innate immune sensors, called pattern recognition receptors, such as TLRs, on immune cells in the tumor microenvironment, which amplifies the inflammatory responses by activating downstream molecular targets such as NF-kB and STAT3. These transcription factors activate genes that control cell survival, proliferation, pro-invasiveness, as well as cytokine and chemokine production (e.g., Bcl-xL, survivin, and c-Myc). Altered TLR expression has been associated with autoimmunity and chronic inflammatory diseases including atherosclerosis, liver disease, and cancer. [39][40][41] Cancer treatment modalities such as surgery, chemotherapy, and radiotherapy can induce local and systemic inflammation due to direct tissue trauma and cancer cell death. Stressed and dying cells (apoptotic or necrotic) shed molecular motifs known as DAMPs, including cell-free nucleic acids (cfDNA, cfRNA, cf-miRNA), mitochondrial DNA (mtDNA), nucleosomes, and exosomes, 42-49 that elicit TLR-mediated pro-inflammatory responses. In addition to immune cells, cancer cells, including breast cancer cells, express TLR receptors. 25,27,40,50 TLR signaling may facilitate metastasis by augmenting tumor cell adhesion and invasion and increasing vascular permeability; 51,52 however, the complex signaling feedback in these axes means the role of TLRs in the events leading to cancer metastasis has yet to be fully elucidated. Overall, since it has been shown that NA DAMPs contribute to cancer progression, we believe the scavenging of these molecules can potentially limit these chronic inflammatory states. 28,[53][54][55] Previously, we demonstrated that PAMAM-G3 inhibits NA DAMP induction of TLR-mediated pancreatic cancer tumor cell invasion in vitro and reduces liver metastasis in an orthotopic immunocompetent syngeneic murine model. 36 Most recently, we demonstrated that PAMAM-G3 is able to reduce lung metastasis in both an intravenous and orthotopic murine model of breast cancer. 56 Here, PAMAM-G3 controlled systemic inflammation by decreasing NA DAMPs, inflam-matory immune cell subtypes, and proinflammatory cytokine levels. Based on these observations, we hypothesized that the NAS-mediated approach to controlling cancer-associated inflammation, migration, and metastasis should be evaluated in the more clinically relevant samples and in a more relevant murine model of breast cancer. Therefore, in the present study, we investigate if PAMAM-G3 treatment limits the ability of chemotherapy-induced TNBC patient DAMPs to induce inflammatory responses and breast cancer cell migration ex vivo and mitigates metastasis in an aggressive, genetically engineered murine model of spontaneous breast cancer.

Breast cancer cell lines strongly express functional TLR9
Several investigations have suggested that the DNA-sensing TLR9 may play a role in the invasive capability of TNBC due to dying tumor cells releasing DNA and activating pro-growth signaling cascades downstream of TLR9 in nearby breast cancer cells. 25,37,50,57 Therefore, we screened a panel of breast cancer cell lines (ER+, TNBC, inflammatory breast cancer, and murine breast cancer cell lines) for TLR9 expression. As shown in Figure 1A, we determined via western blot analysis that several murine and human breast cancer cell lines express higher levels of the DNA-sensing TLR9 compared to the basal expression of TLR9 in HEK293 cells engineered to overexpress human murine TLR4 (HEK293-hTLR4 and HEK293-mTLR4).
nuclear cell lysates were generated from the TNBC cell lines SUM149 and MDA-MB-231 and compared to nuclear cell lysates from HEK293-hTLR9. These cells had been treated with or without a TLR9 agonist (CpG ODN-2006). As shown in Figures 1B-1D, addition of CpG DNA resulted in a significant increase in nuclear levels of NF-kB (measured via an ELISA probe of the RelA p65 subunit of the NF-kB protein complex), demonstrating that the TLR9 on the cancer cells responds to CpG DNA treatment. In addition, treatment with the NAS PAMAM-G3 mitigates the activation of TLR9 by CpG DNA (Figures 1B-1D). These results were recapitulated in both the TNBC (MDA-MB-231) and TLR9 reporter (HEK293-hTLR9) cell lines ( Figures 1C and 1D). These observations indicate that PAMAM-G3 treatment leads to a reduction in downstream activation of NF-kB through the blocking of TLR9 signaling in these TNBC cells.
Chemotherapy treatment of breast cancer increases DNAcontaining DAMP levels, and NAS treatment counteracts DAMPmediated activation of TLR9 Next, we evaluated if TNBC TLR9-positive cells release their DNA following chemotherapeutic treatment. We quantified the amount of cfDNA generated by two TNBC cell lines, MDA-MB-231 and SUM149, that were treated with a chemotherapy regimen ( To determine if such samples activate TLR9, we employed human TLR9-overexpressing HEK293 reporter cells (HEK293-hTLR9). As expected, treatment of the reporter cells with a known TLR9 agonist, CpG (ODN 2006), led to an increase in TLR9 activation. This activation was reversed by PAMAM-G3 treatment to a similar degree as by a known synthetic oligonucleotide TLR9 inhibitor, ODN A151 (Figure 2D). Next, we evaluated the effects of PAMAM-G3 treatment on  Figure 2F (SUM149), the TNBC-derived DAMPs are able to activate the TLR9 reporter cells also. Importantly, PAMAM-G3 treatment was able to significantly reduce TLR9 activation by the chemotherapy-derived NA DAMPs released from treated TNBC cells.
Lastly, we evaluated whether clinically relevant NA DAMPs present in post-neoadjuvant chemotherapy (post-NAC) plasma collected from breast cancer patients activate TLR9 and if NAS treatment can mitigate such activation. Again, using HEK293-hTLR9 reporter cells, we screened a small panel of four breast cancer post-NAC patient plasma samples to determine if they can activate TLR9. We observed that both post-NAC TNBC patient plasma and luminal B (ER + /PR À /HER2 + ) patient plasma strongly induced TLR9 signaling ( Figure 2G; Figure S1). Next, we evaluated whether the NAS PAMAM-G3 could impede such activation. As shown in Figure 2G, in all four patients, PAMAM-G3 treatment reduced the ability of breast cancer patient plasma to activate the TLR9 pathway as measured by NF-kB activation. These results indicate that post-NAC patient plasma contains NA DAMPs that can induce TLR9 activation, and this proinflammatory process can be counteracted by PAMAM-G3 treatment.
Chemotherapy-derived CM and post-NAC breast cancer patient plasma induce TNBC invasion, and NAS treatment mitigates this pathological process Once we determined that chemotherapy-derived TNBC CM and post-NAC breast cancer patient plasma were rich in NA DAMPs that activate TLR9 ( Figure 2) and that TNBC cells lines such as SUM149 and MDA-MB-231 express functional TLR9 (Figure 1), we hypothesized that in vitro treatment of TNBC cells with a TLR9 agonist would induce a pro-invasive phenotype. Invasive potential was measured using a Transwell-Matrigel invasion assay as detailed in Figure 3A. We tested SUM149 and MDA-MB-231 cell invasion potential when incubated with CpG DNA or TNBC chemotherapyderived CM in a Transwell-Matrigel invasion assay. As shown in Figures Figure S2).
Next, we examined if TNBC post-NAC patient plasma induces a similar pro-invasive response in SUM149 cells. In addition, we wanted to evaluate the effect of PAMAM-G3 treatment in this setting. SUM149 cells were incubated in the presence of post-NAC TNBC patient plasma or pooled normal human plasma with or without PAMAM-G3, and invasive potential was measured using the Transwell-Matrigel invasion assay ( Figures 3D and 3G). Post-NAC TNBC patient plasma induced cancer invasion, while normal plasma did not. PAMAM-G3 was once again able to largely abrogate the TNBC patient plasma-mediated increase in TNBC cell invasion. Similar results were obtained when using post-NAC luminal B patient plasma (Figures 3D and 3G). Thus, TLR9-positive TNBC cells are responsive to TLR9 agonists released by chemotherapy-treated TNBC cell lines and breast cancer patients, and NAS treatment can counteract such responses ex vivo.
NAS treatment reduces lung metastasis in a genetically engineered murine model of spontaneous breast cancer Given the effects of PAMAM-G3 in reducing NA DAMPs signaling and cancer cell invasion, and since we had previously shown that the NAS could reduce lung metastatic burden in artificial orthotopic experimental murine models, 56 we next sought to evaluate PAMAM-G3's efficacy in a more clinically relevant spontaneous murine model of breast cancer. We utilized the aggressive mouse mammary tumor virus-poly-oma middle T antigen mouse strain (MMTV-PyMT; FVB background), which contains the polyoma virus middle T-antigen as a transgene expressed from the MMTV LTR promoter. Hemizygous females undergo a course of mammary gland neoplasia that resembles aggressive human breast cancer pathogenesis 62-64 and ultimately develop lung metastases ( Figure S3). As shown in Figure 4A, hemizygous MMTV-PyMT females develop breast cancer in multiple mammary glands, but their transgenenegative sisters (identified via PCR genotyping as seen in Figure S4) do not. Hemizygous female mice were treated with either saline or PAMAM-G3 (2Â/week, 20 mg/kg, intraperitoneal (i.p.) injections) from 5 weeks of age to 12 weeks of age, then sacrificed.
Primary tumor burden was evaluated by measuring the gross mass of excised mammary glands from each treated animal ( Figure 4C). PAMAM-G3 treatment did have a modest but significant reductive effect on the primary tumor burden in the mammary glands. However, histological analyses revealed a marked reduction in metastatic lung lesions in the lungs of PAMAM-G3-treated mice compared to saline-treated mice ( Figure 4B). This observation was confirmed by significantly lower lung masses and metastatic lung surface areas (Figures 4D and 4E) in PAMAM-G3-treated mice as compared to salinetreated mice.

DISCUSSION
In this study, we found that treatment with chemotherapy leads to an increase in NA DAMP release from breast cancer cells in culture and into circulation in patients with breast cancer. Moreover, CM from TNBC cells treated with chemotherapy and plasma from breast cancer patients treated with chemotherapy robustly stimulate human TLR9 activation and signaling in reporter cells and induce a pro-invasive phenotype in human TNBC cells. Both of these NA DAMPmediated effects can be mitigated by PAMAM-G3 treatment. Importantly, PAMAM-G3 treatment significantly decreased lung metastasis in a genetically engineered mouse model of spontaneous breast cancer. The decrease in lung metastasis observed after PAMAM-G3 treatment in such an aggressive breast cancer mouse model suggests that effective blocking of NA DAMP-induced inflammation with a NAS could be a valuable therapeutic approach to limit breast cancer metastasis, particularly when utilized in combination with the current standard-of-care chemotherapy, which, as demonstrated in this study, may itself promote metastatic spread. Use of a NAS (such as PAMAM-G3) as an early adjuvant therapy may be an effective approach to help limit breast cancer metastasis.
Systemic cancer therapies have been reported to induce aberrant inflammation 32,65-67 and play a role in the enhanced metastatic behavior seen in the treatment-resistant clones that survive the initial therapeutic culling. 68,69 Therefore, the characterization of diseasespecific DAMPs that contribute to tumor progression and metastasis is of clinical importance (bearing in mind that a variety of DAMPs can activate TLR and other innate immune system signaling cascades such as HMGB-1 and histone proteins). Unfortunately, the redundant nature of DAMPs and pattern recognition receptors (PRRs) likely means that defining a complete mechanistic understanding of their actions in a complex tumor-immunocompetent setting will be challenging. 70 Nevertheless, the potential to broadly block NA DAMP-induced inflammation with a NAS may be of significant therapeutic benefit, since some tumors tend to develop drug resistance via redundant signaling nodes, clonal heterogeneity, and immunomodulation-thus making their treatment unlikely to depend on one concomitant signaling pathway. Nonetheless, it is likely that a NAS-based protocol that targets inflammation-induced metastasis will require combination with other therapeutic agents for the clinical management of various malignancies. [71][72][73] Previously, we have observed that PAMAM-G3 treatment can limit metastasis in syngeneic mouse models of pancreatic and breast cancer metastasis. 36,56 The studies described herein build upon these studies that found that PAMAM-G3 treatment could limit artificial metastasis from the spleen to the liver (ectopic pancreatic cancer model) or mammary fat pad to the lung (orthotopic breast cancer model).
Thus, these results significantly extend and corroborate prior studies and advance the field by moving it from artificial, non-spontaneous breast cancer animal models into a highly aggressive spontaneous cancer mouse model and moreover demonstrate that breast cancer patient samples induce similar NA DAMP-engendered activities in cell culture, which can be mitigated by NAS treatment. This study provides additional evidence about the importance of TLR9 signaling in mediating invasion and metastasis of breast cancer and describes an innovative chemical biology approach using nucleic-acid-binding polymers to scavenge such TLR9 agonists and mitigate such pathological effects. By targeting a phenotype that has been implicated in various cancer contexts, namely persistent nucleic acid DAMPinduced cell activation and inflammation, the NAS approach impedes an important signaling axis in difficult-to-treat inflammation-dependent malignancies. Given the lack of effective treatments for metastatic TNBC, this project highlights a potentially significant complementary treatment that could be utilized to reduce the mortality seen in this breast cancer patient population and facilitate the development of a novel class of anti-metastatic therapies using NASs. after which the absorbance at 655 nm was measured using a Spectramax i3 plate reader (Molecular Devices).

Quantification of cfDNA levels
Total DNA was isolated from plasma using the DNA Blood Mini Kit (QIAGEN) or from therapy-treated CM supernatant from TNBC cell lines and quantified using the Quant-iT PicoGreen dsDNA Staining Kit (Thermo Fisher Scientific).
Analysis of nuclear NF-kB translocation SUM149 cells were plated at $2 Â 10 6 cells per plate in T-75 flasks overnight. The cells were then treated with serum-containing media alone, CpG ODN 2006 (10 mM), and PAMAM-G3 (50 mg/mL) for 4 h. Cell nuclear lysates were then isolated using a nuclear isolation kit (Abcam) according to the manufacturer's guidelines. The protein content of each nuclear extract was quantified and standardized using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). The nuclear extract was then used to quantify nuclear translocation of NF-kB using the p65 RelA subunit ELISA kit (Abcam) according to the manufacturer's guidelines.

Western blot
Protein lysates were prepared by using Pierce RIPA lysis and extraction buffer (Thermo Fisher Scientific) containing Halt Protease and Phosphatase inhibitors (1Â) on 80%-90% confluent T-75 flasks of each cell line. 250 mL of lysis buffer was added to the cells, and then they were scraped into clean microcentrifuge tubes and placed on ice for 10-15 min. Tubes were then agitated at 4 C for 15 min. Then the tubes were centrifuged for 30 min at 4 C. The supernatant was then transferred to fresh centrifuge tubes, and the protein content was quantified and standardized using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Immunoblotting was carried out using standard protocols with R30 mg of total protein mixed with Laemmli sample buffer (Bio-Rad) in each well of the Mini-PROTEAN TGX gels (Bio-Rad). Gels were run at 220-250 V for 30-40 min. Activate LF-PVDF membranes in high-performance liquid chromatography (HPLC)-grade methanol for 5 min. Gels were then transferred to LF-PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Antibodies in 3% w/v BSA in 1Â TBST were then used to probe the membranes. The following membranes were purchased from Cell Signaling Technology (b-actin, GAPDH) or Santa Cruz Biotechnology (TLR4, TLR9). Blots were then visualized on a Bio-Rad ChemiDoc Imager.

Transwell-Matrigel invasion chamber assays
Transwell-Matrigel inserts and 24-well plates were purchased from Corning, and invasion assays were performed according to the manufacturer's instructions. The invasion inserts were first incubated with serum-free media in the top and bottom chambers at 37 C for at least 2-4 h to allow the Matrigel to equilibrate and solidify before the addition of the TNBC cells and their various treatment conditions (CM, agonists, PAMAM-G3). The human TNBC SUM149 cell line was trypsinized, inactivated with serum-rich media, centrifuged for 5 min at 300 Â g, and then resuspended in serum-free media at the correct dilution. After aspiration of serum-free media from the equilibrated invasion chambers, SUM149 cells were plated into the top chamber at a density of 35,000 cells/well (500 mL final volume) in the presence of serum-free media alone or various combinations of agonists and/or PAMAM-G3 (25 or 50 mg/mL). TLR agonists tested included CpG ODN-2006 (10 mM), TNBC patient plasma, or normal human plasma (25 mL). A total of 750 mL of complete growth media was added to the bottom chambers to create a nutrient gradient. All conditions were tested in at least duplicate. The plated invasion chambers were incubated at 37 C for 22-26 h, after which time media was aspirated from the top chambers.
The top chambers were then removed and placed into a new 24-well plate preloaded with 1 mL of 10% formaldehyde per well to fix the invaded cells on the bottom surface of the chamber. After fixation for 10 min, the top chambers were then removed from the formaldehyde and placed into a new 24-well plate preloaded with 1 mL of PBS (without divalent cations) per well for 1 min for washing purposes. The top chambers were then placed onto absorbent pads, and residual PBS was wiped from the inside of the top chambers using a cotton swab. 10 mL of crystal violet solution (5% w/v crystal violet, 25% v/ v methanol) was added to the membranes and allowed to stain for 5-10 min. Crystal violet was purchased from Sigma-Aldrich. The chambers were then washed thoroughly in deionized water, and residual crystal violet was wiped from the inside of the chambers using a cotton swab. The membranes were then imaged using an inverted light microscope (Olympus IX50) at 10Â magnification. A total of 6-8 random image view fields of each membrane were taken using a digital eyepiece camera (Dino-Eye AM7023B). The cells in the images were then counted using an ImageJ algorithm unique for each cell line (https://imagej.net/Particle_Analysis).

Genetically engineered breast cancer murine model
All animal experiments were performed in compliance with the Duke Institutional Animal Care and Use Committee (IACUC) protocols and housed in an animal facility at Duke University (Durham, NC, USA). Male MMTV-PyMT transgenic mice (stock no: 002374) and female FVB/NJ mice (stock no: 001800) were purchased from The Jackson Laboratory. A breeding colony was established and maintained by crossing male transgenics to congenic FVB female mice. PCR analysis of DNA prepared from a tail snip was used to identify carriers possessing the MMTV-PyMT transgene ( Figure S4). A positive control fragment (200 bp) was amplified in all samples, and, as expected, a transgene fragment (556 bp) amplified in approximately half of the samples. Starting 5 weeks post birth, hemizygous MMTV-PyMT females were treated intraperitoneally twice per week with either PAMAM-G3 (20 mg/kg) or saline until mice were euthanized at 12 weeks of age. Initially, each treatment group contained 15 mice each, and there were no associated PAMAM-G3 deaths. One mouse from each treatment group was removed due to having outlier measurements beyond 3 standard deviations of the mean of said treatment group. An additional PAMAM-G3 mouse was excluded due to having red lungs of double the usual mass, and histological evaluation indicating evidence of a hemorrhage. www.moleculartherapy.org Mammary glands and lungs were harvested from each mouse for assessment of gross organ mass. Lungs were fixed in formalin, and hematoxylin and eosin (H&E) staining was performed on three lung sections per animal, with 100 mm between slices (HistoWiz). With blinding as to treatment status, each metastatic lesion was identified, and then associated surface area determined based on the section where the lesion was largest using ImageJ.

Quantification and statistical analysis
For all assays and experiments, statistical comparison between treatment groups was first tested for normality via the Shapiro-Wilk test. If normal, the groups were then compared via a two-tailed Student's t test or an ANOVA with an appropriate post hoc test. If the data were not normal, then they were log-transformed and re-tested for normality. If normal, data were analyzed by a two-tailed Student's t test or an ANOVA with a Holm-Sídák or Tukey's test. If not normal, the data were then tested with the non-parametric Fisher's exact test. All data were plotted and statistically analyzed using GraphPad Prism (version 6.0 or 8.3) or JMP software. All data were presented as the mean ± standard error determined from 3-5 technical replicates. All biological experiments were performed at least twice.