Kathleen Burke

Second harmonic generation reveals matrix alterations during breast tumor progression

Abstract. Alteration of the extracellular matrix in tumor stroma influences efficiency of cell locomotion away from the primary tumor into surrounding tissues and vasculature, thereby affecting metastatic potential. We study matrix changes in breast cancer through the use of second harmonic generation (SHG) of collagen in order to improve the current understanding of breast tumor stromal development. Specifically, we utilize a quantitative analysis of the ratio of forward to backward propagating SHG signal (F/B ratio) to monitor collagen throughout ductal and lobular carcinoma development. After detection of a significant decrease in the F/B ratio of invasive but not in situ ductal carcinoma compared with healthy tissue, the collagen F/B ratio is investigated to determine the evolution of fibrillar collagen changes throughout tumor progression. Results are compared with the progression of lobular carcinoma, whose F/B signature also underwent significant evolution during progression, albeit in a different manner, which offers insight into varying methods of tissue penetration and collagen manipulation between the carcinomas. This research provides insights into trends of stromal reorganization throughout breast tumor development.

Enzymatically-responsive pro-angiogenic peptide-releasing poly(ethylene glycol) hydrogels promote vascularization in vivo

Therapeutic angiogenesis holds great potential for a myriad of tissue engineering and regenerative medicine approaches. While a number of peptides have been identified with pro-angiogenic behaviors, therapeutic efficacy is limited by poor tissue localization and persistence. Therefore, poly(ethylene glycol) hydrogels providing sustained, enzymatically-responsive peptide release were exploited for peptide delivery. Two pro-angiogenic peptide drugs, SPARC113 and SPARC118, from the Secreted Protein Acidic and Rich in Cysteine, were incorporated into hydrogels as crosslinking peptides flanked by matrix metalloproteinase (MMP) degradable substrates. In vitro testing confirmed peptide drug bioactivity requires sustained delivery. Furthermore, peptides retain bioactivity with residual MMP substrates present after hydrogel release. Incorporation into hydrogels achieved enzymatically-responsive bulk degradation, with peptide release in close agreement with hydrogel mass loss and released peptides retaining bioactivity. Interestingly, SPARC113 and SPARC118-releasing hydrogels had significantly different degradation time constants in vitro (1.16 and 8.77×10(-2) h(-1), respectively), despite identical MMP degradable substrates. However, upon subcutaneous implantation, both SPARC113 and SPARC118 hydrogels exhibited similar degradation constants of ~1.45×10(-2) h(-1), and resulted in significant ~1.65-fold increases in angiogenesis in vivo compared to controls. Thus, these hydrogels represent a promising pro-angiogenic approach for applications such as tissue engineering and ischemic tissue disorders.

The antidepressant desipramine and α2-adrenergic receptor activation promote breast tumor progression in association with altered collagen structure.

Emotional stress activates the sympathetic nervous system (SNS) and release of the neurotransmitter norepinephrine to promote breast tumor pathogenesis. We demonstrate here that the metastatic mammary adenocarcinoma cell line 4T1 does not express functional adrenergic receptors (AR), the receptors activated by norepinephrine, yet stimulation of adrenergic receptor in vivo altered 4T1 tumor progression in vivo. Chronic treatment with the antidepressant desipramine (DMI) to inhibit norepinephrine reuptake increased 4T1 tumor growth but not metastasis. Treatment with a highly selective α2-adrenergic receptor agonist, dexmedetomidine (DEX), increased tumor growth and metastasis. Neither isoproterenol (ISO), a β-AR agonist, nor phenylephrine, an α1-AR agonist, altered tumor growth or metastasis. Neither DMI- nor DEX-induced tumor growth was associated with increased angiogenesis. In DMI-treated mice, tumor VEGF, IL-6, and the prometastatic chemokines RANTES, M-CSF, and MIP-2 were reduced. Tumor collagen microstructure was examined using second harmonic generation (SHG), a nonabsorptive optical scattering process to highlight fibrillar collagen. In DMI- and DEX-treated mice, but not ISO-treated mice, tumor SHG was significantly altered without changing fibrillar collagen content, as detected by immunofluorescence. These results demonstrate that α2-AR activation can promote tumor progression in the absence of direct sympathetic input to breast tumor cells. The results also suggest that SNS activation may regulate tumor progression through alterations in the extracellular matrix, with outcome dependent on the combination of adrenergic receptor activated. These results underscore the complexities underlying SNS regulation of breast tumor pathogenesis, and suggest that the therapeutic use of adrenergic receptor blockers, tricyclic antidepressants, and adrenergic receptor agonists must be approached cautiously in patients with breast cancer. Cancer Prev Res; 6(12); 1262–72. ©2013 AACR.

Temporally tunable, enzymatically responsive delivery of proangiogenic peptides from poly(ethylene glycol) hydrogels.

Proangiogenic drugs hold great potential to promote reperfusion of ischemic tissues and in tissue engineering applications, but efficacy is limited by poor targeting and short half-lives. Methods to control release duration or provide enzymatically responsive drug delivery have independently improved drug efficacy. However, no material has been developed to temporally control the rate of enzymatically responsive drug release. To address this void, hydrogels are developed to provide sustained, tunable release of Qk, a proangiogenic peptide mimic of vascular endothelial growth factor, via tissue-specific enzymatic activity. After confirmation that sustained delivery of Qk is necessary for proangiogenic effects, a variety of previously identified matrix metalloproteinase (MMP)-degradable linkers are used to tether Qk to hydrogels. Of these, three (IPES↓LRAG, GPQG↓IWGQ, and VPLS↓LYSG) show MMP-responsive peptide release. These linkers provide tunable Qk release kinetics, with rates ranging from 1.64 to 19.9 × 10(-3) h(-1) in vitro and 4.82 to 8.94 × 10(-3) h(-1) in vivo. While Qk is confirmed to be bioactive as released, hydrogels releasing Qk fail to induce significant vascularization in vivo after one week, likely due to the use of nonenzymatically degradable hydrogels. While Qk is the focus of this study, the approach could easily be adapted to control the delivery of a variety of therapeutic molecules.

Stromal matrix metalloprotease-13 knockout alters Collagen I structure at the tumor-host interface and increases lung metastasis of C57BL/6 syngeneic E0771 mammary tumor cells

BackgroundMatrix metalloproteases and collagen are key participants in breast cancer, but their precise roles in cancer etiology and progression remain unclear. MMP13 helps regulate collagen structure and has been ascribed largely harmful roles in cancer, but some studies demonstrate that MMP13 may also protect against tumor pathology. Other studies indicate that collagen’s organizational patterns at the breast tumor-host interface influence metastatic potential. Therefore we investigated how MMP13 modulates collagen I, a principal collagen subtype in breast tissue, and affects tumor pathology and metastasis in a mouse model of breast cancer.MethodsTumors were implanted into murine mammary tissues, and their growth analyzed in Wildtype and MMP13 KO mice. Following extraction, tumors were analyzed for collagen I levels and collagen I macro- and micro-structural properties at the tumor-host boundary using immunocytochemistry and two-photon and second harmonic generation microscopy. Lungs were analyzed for metastases counts, to correlate collagen I changes with a clinically significant functional parameter. Statistical analyses were performed by t-test, analysis of variance, or Wilcoxon-Mann–Whitney tests as appropriate.ResultsWe found that genetic ablation of host stromal MMP13 led to: 1. Increased mammary tumor collagen I content, 2. Marked changes in collagen I spatial organization, and 3. Altered collagen I microstructure at the tumor-host boundary, as well as 4. Increased metastasis from the primary mammary tumor to lungs.ConclusionsThese results implicate host MMP13 as a key regulator of collagen I structure and metastasis in mammary tumors, thus making it an attractive potential therapeutic target by which we might alter metastatic potential, one of the chief determinants of clinical outcome in breast cancer. In addition to identifying stromal MMP13 is an important regulator of the tumor microenvironment and metastasis, these results also suggest that stromal MMP13 may protect against breast cancer pathology under some conditions, a finding with important implications for development of chemotherapies directed against matrix metalloproteases.

Second-harmonic generation scattering directionality predicts tumor cell motility in collagen gels

Abstract. Second-harmonic generation (SHG) allows for the analysis of tumor collagen structural changes throughout metastatic progression. SHG directionality, measured through the ratio of the forward-propagating to backward-propagating signal (F/B ratio), is affected by collagen fibril diameter, spacing, and disorder of fibril packing within a fiber. As tumors progress, these parameters evolve, producing concurrent changes in F/B. It has been recently shown that the F/B of highly metastatic invasive ductal carcinoma (IDC) breast tumors is significantly different from less metastatic tumors. This suggests a possible relationship between the microstructure of collagen, as measured by the F/B, and the ability of tumor cells to locomote through that collagen. Utilizing in vitro collagen gels of different F/B ratios, we explored the relationship between collagen microstructure and motility of tumor cells in a “clean” environment, free of the myriad cells, and signals found in in vivo. We found a significant relationship between F/B and the total distance traveled by the tumor cell, as well as both the average and maximum velocities of the cells. Consequently, one possible mechanism underlying the observed relationship between tumor F/B and metastatic output in IDC patient samples is a direct influence of collagen structure on tumor cell motility.

Using second harmonic generation to predict patient outcome in solid tumors

BackgroundOver-treatment of estrogen receptor positive (ER+), lymph node-negative (LNN) breast cancer patients with chemotherapy is a pressing clinical problem that can be addressed by improving techniques to predict tumor metastatic potential. Here we demonstrate that analysis of second harmonic generation (SHG) emission direction in primary tumor biopsies can provide prognostic information about the metastatic outcome of ER+, LNN breast cancer, as well as stage 1 colorectal adenocarcinoma.MethodsSHG is an optical signal produced by fibrillar collagen. The ratio of the forward-to-backward emitted SHG signals (F/B) is sensitive to changes in structure of individual collagen fibers. F/B from excised primary tumor tissue was measured in a retrospective study of LNN breast cancer patients who had received no adjuvant systemic therapy and related to metastasis-free survival (MFS) and overall survival (OS) rates. In addition, F/B was studied for its association with the length of progression-free survival (PFS) in a subgroup of ER+ patients who received tamoxifen as first-line treatment for recurrent disease, and for its relation with OS in stage I colorectal and stage 1 lung adenocarcinoma patients.ResultsIn 125 ER+, but not in 96 ER-negative (ER-), LNN breast cancer patients an increased F/B was significantly associated with a favorable MFS and OS (log rank trend for MFS: p = 0.004 and for OS: p = 0.03). On the other hand, an increased F/B was associated with shorter PFS in 60 ER+ recurrent breast cancer patients treated with tamoxifen (log rank trend p = 0.02). In stage I colorectal adenocarcinoma, an increased F/B was significantly related to poor OS (log rank trend p = 0.03), however this relationship was not statistically significant in stage I lung adenocarcinoma.ConclusionWithin ER+, LNN breast cancer specimens the F/B can stratify patients based upon their potential for tumor aggressiveness. This offers a “matrix-focused” method to predict metastatic outcome that is complementary to genomic “cell-focused” methods. In combination, this and other methods may contribute to improved metastatic prediction, and hence may help to reduce patient over-treatment.

The Use of Second Harmonic Generation to Image the Extracellular Matrix During Tumor Progression

Abstract Metastasis is the leading cause of cancer mortality, resulting from changes in the tumor microenvironment which increases tumor cell migration, dispersal to distant organs, and subsequent survival. This is accompanied by changes in tumor collagen which may allow cells to travel more efficiently away from a primary tumor and invade the surrounding tissue. Second Harmonic generation (SHG) is an intrinsic optical signal that has expanded our understanding of collagen evolution throughout tumor progression. This article addresses current research into tumor progression using SHG, as well as the future prospects of using SHG to advance our understanding of the tumor microenvironment.

Second-harmonic generation reveals a relationship between metastatic potential and collagen fiber structure

Second Harmonic Generation (SHG) of collagen signals allows for the analysis of collagen structural changes throughout metastatic progression. The directionality of coherent SHG signals, measured through the ratio of the forward-propagating to backward propagating signal (F/B ratio), is affected by fibril diameter, spacing, and order versus disorder of fibril packing within a fiber. As tumors interact with their microenvironment and metastasize, it causes changes in these parameters, and concurrent changes in the F/B ratio. Specifically, the F/B ratio of breast tumors that are highly metastatic to the lymph nodes is significantly higher than those in tumors with restricted lymph node involvement. We utilized in vitro analysis of tumor cell motility through collagen gels of different microstructures, and hence different F/B ratios, to explore the relationship between collagen microstructures and metastatic capabilities of the tumor. By manipulating environmental factors of fibrillogenesis and biochemical factors of fiber composition we created methods of varying the average F/B ratio of the gel, with significant changes in fiber structure occurring as a result of alterations in incubation temperature and increasing type III collagen presence. A migration assay was performed using simultaneous SHG and fluorescent imaging to measure average penetration depth of human tumor cells into the gels of significantly different F/B ratios, with preliminary data demonstrating that cells penetrate deeper into gels of higher F/B ratio caused by lower type III collagen concentration. Determining the role of collagen structure in tumor cell motility will aid in the future prediction metastatic capabilities of a primary tumor.